Binding of magnesium ions to cell walls of Bacillus subtilis W23 containing teichoic acid or teichuronic acid.

When grown in a chemostat under various nutritional conditions, cells of Bacillus subtilis W23 produce walls containing teichoic acid or teichuronic acid. The binding of Mg2+ to these walls and to the isolated anionic polymers in solution was measured by equilibrium dialysis. In solution the ribitol teichoic acid bound Mg2+ in the molar ratio Mg2+/P=1:1 with an apparent association constant (Kassoc.) of 0.61 X 10(3)M-1, and the teichuronic acid bound Mg2+ in the ratio Mg2+/CO2-=1.1, Kassoc.=0.3 X 10(3)M-1. Cell walls containing teichuronic acid exhibited closely similar binding properties to those containing teichoic acid; in both cases Mg2+ was bound in the ratio Mg/P or Mg/CO2- of 0.5:1 and with a greater affinity than displayed by the isolated polymers in solution. It was concluded that Mg2+ ions are bound bivalently between anionic centres in the walls and that the incorporation of teichoic acid or teichuronic acid into the walls gives rise to similar ion-binding and charged properties. The results are discussed in relation to the possible functions of anionic polymers in cell walls.     

Role of teichuronic acid in Bacillus licheniformis: defective autolysis due to deficiency of teichuronic acid in a novobiocin-resistant mutant.

nov-12, a novobiocin-resistant mutant of Bacillus licheniformis ATCC 9945, grows as long chains of cells, a characteristic of autolytic-deficient (Lyt-) mutants. Isolated walls from nov-12 autolyzed at a rate equal to 5% of that displayed by wild-type walls, thus confirming the Lyt- phenotype. Protein-free nov-12 walls displayed marked resistance to, and also failure to bind, added autolysin solubilized from wild-type walls. Comparison of isolated cell walls revealed a deficiency in teichuronic acid in the mutant. Lesser differences were observed in walls of this strain, including a reduction in galactose, an increase in the proportion of peptidoglycan, and small quantitative differences in peptidoglycan composition though the proportions of protein and teichoic acid were similar in walls of both strains. Autolytic sensitivity was studied in walls in which protein, teichoic acid, and teichuronic acid were removed successively by selective extraction procedures. Autolysis of wild-type walls was unaffected by removal or protein or teichoic acid, but teichuronic acid removal rendered wild-type walls as insensitive to autolysis as mutant walls had been throughout. Therefore, in this mutant, deficiency in teichuronic acid alone leads to the Lyt- phenotype and, hence, activity and binding of autolysin(s) are dependent upon teichuronic acid but not teichoic acid. Also, the potential rate of autolysis of cell walls in this organism was correlated with the proportion of teichuronic acid in the wall. The possible significance of these findings with respect to control of autolysis and cell separation is discussed.     

Structure of a polysaccharide containing galactose and galacturonic acid from Rhizobium meliloti. Characterization and partial purification of a 2-O-methyltransferase

The teichuronic acid type polysaccharide found in Rhizobium meliloti which is associated with sensitivity to phage 16B and is formed in the inner membranes from UDP-galactose and UDP-galacturonic acid (Ugalde, R. A., Coira, J. A., and Brill, W. J. (1986) J. Bacteriol. 168, 270-275) has been studied further. Results of acid hydrolysis, periodate oxidation, and borohydride reduction show that this polysaccharide contains the repetitive unit -galacturonosyl(1-3)galactosyl(1-4-). A soluble enzyme was found to catalyze the transfer of methyl groups from S-adenosylmethionine to position 2 of the galacturonosyl residue. The enzyme requires Mn2+ or Mg2+, its pH optimum is 8.2, and the apparent Km for S-adenosylmethionine is 2.7 microM. The teichuronic acid type polysaccharide bound to a trichloroacetic acid-insoluble cell residue is a substrate for the methyltransferase; however, the polysaccharide released from the trichloroacetic acid-insoluble portion by mild acid treatment is no longer methylated. Other soluble galacturonic acid-containing polysaccharides were not used as substrates. The methyltransferase and the polysaccharide acceptor are both found in R. meliloti strain 102F51. Spontaneously arising mutants resistant to phage 16B do not form teichuronic acid but are transferase-positive. Other strains of R. meliloti as well as Agrobacterium tumefaciens and Escherichia coli cells do not form teichuronate and have no transferase.     

Function of cell wall teichoic acid in thermally injured Staphylococcus aureus.

Thermally injured cells of Staphylococcus aureus lack the ability to grow on tryptic soy agar containing 7.5% NaCl. This injury phenomenon was examined in three strains of S. aureus: MF-31; H (Str); and, isolated from H (Str), 52A5, a mutant which lacks teichoic acid in the cell wall. Temperatures for sublethal heat treatment were selected to produce maximum injury with minimum death for each strain. Examination of isolated cell walls showed that magnesium was lost from the wall during heating, and that the degree of cell injury was accentuated when magnesium ions were either removed from or made unavailable to the cell. S. aureus 52A5 was more heat sensitive than its parent strain. Cells containing higher levels of wall teichoic acid generally showed less injury than normal cells. Cells with the weaker cation-binding polymer, teichuronic acid, in the cell wall generally showed greater injury. These data suggest that cell wall teichoic acid of S. aureus aids in the survival of the cell by the maintenance of an accessible surface pool of magnesium.     

Chemical and ultrastructural examination of lead-induced morphological convertants of Bacillus subtilis.

Actively-growing Bacillus subtilis 168 cells, exposed to lead nitrate, had only slightly decreased ability to bind the divalent cation magnesium. The nature and quantity of the major cell wall metal binding components, teichoic and teichuronic acids, and the carbohydrate constituents of the peptidoglycan remained relatively constant. Purified cell walls, isolated from cells exposed to lead for 6 and 13 days, retained 9.9 micrograms Pb+2 and 3.5 micrograns Pb+2/mg cell wall, respectively. The occurrence of this lead in the isolated cell wall did not interfere with its Mg2+-binding capacity. While cationic binding properties, growth rate and cell yield indicated non-interference from lead, light and electron microscopic studies clearly demonstrated morphological alterations in approx. 30--50% of the lead treated cells. These alterations included the conversion to irregular spherical forms, some of which contained thickened cell walls. These anomalies are virtually identical to those reported for mutants of this bacterium derived via the introduction of classical mutagens. Protoplasts, similar to those produced by antibiotic and enzymatic treatments, were also present in the lead-treated cells. Although the most tenable explanation appeared to be cellular mutations, the morphological convertants appear to be derived from lead's interference with cell wall biosynthesis process and/or the assemblage of cell wall subunits.     

Use of bacteriophage-resistant mutants to study the nature of the bacteriophage receptor site of Staphylococcus aureus

Bacteriophage-resistant strains of Staphylococcus aureus H were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Cell walls isolated from about half of these resistant strains were incapable of inactivating phages and were shown to lack N-acetyl-d-glucosamine (GlcNAc) in their cell wall teichoic acid. Apart from the lack of GlcNAc, two of these mutant strains were deficient in cell wall phosphorus and ester-linked d-alanine. These two strains were also found to be resistant to both phage K and a host-range mutant isolated from the parent phage. These two phages could lyse the other phage-resistant mutants which lacked GlcNAc in their teichoic acid. Cell walls from the remaining phage-resistant mutant strains did inactivate phages and were found to have normal cell wall teichoic acid. Although GlcNAc in teichoic acid was required for phage inactivation, no difference in phage inactivation ability was detected with cell walls isolated from strains of S. aureus having exclusively alpha- or exclusively beta-linked GlcNAc in their cell wall teichoic acid.     

Immunochemical identification of the major cell surface agglutinogen of Acinetobacter calcoaceticus RAG-92

The immunochemical and immunocytochemical characteristics of three Acinetobacter calcoaceticus RAG strains were compared in order to clarify the relationship between antibody-induced agglutination and the production of polyanionic extracellular emulsifier (termed emulsan). In addition to the parent, RAG-92, two mutant strains were examined: (1) a non-agglutinating emulsan-producer (AB15), and (2) an agglutinating mutant (16TLU) defective in the production of emulsan. A combined genetic-immunochemical approach was employed. This included the comparison of crossed immunoelectrophoresis patterns of parent and mutant supernates and the effect of absorption of anti-whole cell antiserum with mutant cells. In addition, agglutinability and competition studies were performed as well as electron microscopic cytochemistry. The results demonstrated that three major antigenic components were associated with the cell surface and the supernate. Mutant cells were altered both in their cell surface properties and in their extracellular products. One antigenic component, termed component C3, was the major cell surface agglutinogen; this component was absent in non-agglutinating mutants. Component C3 may be identical with or attached to the 300 nm projections on the parent cell surface, but it is not directly related to the presence of emulsan. It appears that emulsan plays little or no role in the phenomenon of antibody-induced agglutination of this organism.     

Loss of alkalophily in cell-wall-component-defective mutants derived from alkalophilic Bacillus C-125. Isolation and partial characterization of the mutants.

The cells of alkalophilic Bacillus sp. C-125 are shaped by peptidoglycan and enclosed by two acidic polymers (teichuronic acid and teichuronopeptide), which bind to the peptidoglycan. Three kinds of mutant strains defective in these acidic polymers were isolated from the strain C-125. These mutants grow poorly at alkaline pH to extents related to the degree of defect in the polymers, suggesting that these acidic polymers are essential for growth in an alkaline environment. These polymers may diminish penetration of hydroxide ions.     

Studies of intracellular thymidine nucleotides. Relationship between the synthesis of deoxyribonucleic acid and the thymidine triphosphate pool in Escherichia coli K12.

The two types of mutant strains which show resistance to T-even phage infection have been isolated and been shown to have either a higher or lower ratio of dTDP-sugar to dTTP than that of the parent strains. The one with a higher ratio of dTDP-sugar to dTTP than the parents has a large dTDP-sugar pool and small dTTP pool, and a high level of dTDPG pyrophosphorylase activity. The other one, with a lower ratio of dTDP-sugar to dTTP than the parents, has a small dTDP-sugar pool and large dTTP pool, and a low or deficient level of this enzyme activity. They form an entirely mucoid colony in the synthetic agar plate. Mutant cells (Ter-6 and Ter-21) which have deficient dTDPG pyrophosphorylase activity show 2 -- 3 times higher activity of UDPG pyrophosphoyrlase than that of parent cells. The dTDPG pyrophosphorylase-deficient mutants (Ter-15 and Ter-21) have a 3 -- 4 times higher concentration of dTTP and a faster rate of DNA synthesis and cell division than those of parent strains in growth with external thymine. The dTDPG pyrophosphorylase constitutive mutant (Ter-4) has a 0.5 -- 0.33 smaller dTTP pool and a slower rate of DNA synthesis and cell division than those of parent cells grown in the same medium. In the Ter-15 and Ter-21 mutants, the intracellular dTTP-dependent DNA synthesis rapidly disappeared in thymine suboptimal concentration, but the Ter-4 mutant maintained its dTTP-dependent DNA synthesis over a 20 muM concentration of external thymine. In high concentration (100 muM) of external thymidine, the thymidine effects on the intracellular dTTP concentration do not significantly appear in these enzyme-deficient mutants (Ter-15 and Ter-21). Also, the concentration of intracellular dTTP in the cell growth with external thymidine is 2.5 times greater than that with external thymine in these enzyme-deficient mutants (Ter-15 and Ter-21).     

The cell wall of Bacillus licheniformis N.C.T.C. 6346. Linkage between the teichuronic acid and mucopeptide components

1. After extraction of teichoic acid from cell walls of Bacillus licheniformis with dilute alkali, the insoluble residue contains the teichuronic acid and mucopeptide components and a small amount of residual phosphorus. 2. A complex of teichuronic acid and a part of the mucopeptide was isolated from the soluble fraction obtained by lysozyme treatment of alkali extracted walls. 3. Small-molecular-weight mucopeptide fragments, not containing teichuronic acid, are obtained from the soluble fraction in yields similar to those obtained after treatment of whole walls or acid-extracted walls with lysozyme. 4. The covalent linkages between teichuronic acid and mucopeptide are broken by treatment with dilute acid. The release of teichuronic acid chains is accompanied by the hydrolysis of N-acetylgalactosaminide linkages and the exposed N-acetylgalactosamine residues form chromogen under very mild conditions, indicating that they are substituted on C-3. 5. The initial rate of formation of reactive N-acetylgalactosamine residues during mild acid hydrolysis is parallel to the rate of extraction under the same conditions of teichuronic acid from alkali-treated insoluble walls, and to the rate of acid hydrolysis of glucose 1-phosphate. 6. The results suggest that the teichuronic acid chains are attached through reducing terminals of N-acetylgalactosamine residues to phosphate groups in the mucopeptide. 7. Muramic acid phosphate was isolated from the insoluble mucopeptide remaining after extraction of walls with dilute alkali followed by dilute acid.     

Comparative in vivo nitrogen-15 nuclear magnetic resonance study of the cell wall components of five Gram-positive bacteria.

The proton-decoupled 9.12 MHz 15N NMR spectra of 15N-labeled Bacillus subtilis, Bacillus licheniformis, Staphylococcus auresu, Streptococcus faecalis, and Micrococcus lysodeikticus intact cells, isolated cells walls, and cell wall digests have been examined. The general characteristics of Gram-positive bacteria 15N NMR spectra and described and spectral assignments are provided, which allow in vivo 15N NMR to be applied to a wide range of problems in bacterial cell wall research. The qualitative similarity of the intact cell and cell wall spectra found in each bacteria allowed the 15 N resonances observed in the proton broad-band noise-decoupled 15N NMR spectra of intact cells to be assigned to cell wall components. Each of the five Gram-positive bacteria displayed a unique set of cell wall 15N resonances, which reflected variations in the primary structure of peptidoglycans and the amounts of teichoic acid and teichuronic acid in the cell wall, as well as the dynamic properties of the cell wall polymers. Spectral assignments of cell wall 15 N resonances assigned to teichoic D-Ala residues, teichuronic acid and acetamido groups, and peptidoglycan acetamido, amide, peptide, and free amino groups have been made on the basis of specific isotopic labeling and dilution experiments, comparison of chemical shifts to literature values, determination of pH titration shifts, cell wall fractionation experiments, and comparative analysis of the cell wall lysozyme digest spectra in terms of the known primary sequences of peptide chains. All the peptidoglycan 15N peptide resonances observed in the intact cells and isolated cell walls could be accounted for by residues in the bridge or crossbar regions of the peptide chains, which indicated that only the cross-linking groups had a high degree of motional freedom. Thermal- and pH-induced conformational changes around the cross-linking D-Ala residues were detected in the B. licheniformis cell wall lysozyme digest products. Comparison of the proton broad-band noise-decoupled and gated decoupled intact cell and cell wall 15N spectra indicated that broad-band proton decoupling resulted in nulling of cytoplasmic resonances and enhancement of the cell wall resonances by the 15N [1H5 nuclear Overhauser effect.     

Spontaneous mutation conferring the ability to catabolize mannopine in Agrobacterium tumefaciens.

Two nopaline-type strains of Agrobacterium tumefaciens, C58 and T37, as well as strain A136, which is a Ti plasmid-cured derivative of strain C58, gave rise to spontaneous mutants that were able to grow on mannopine. The observation of mutagenesis with strain A136 demonstrated that the ability to acquire this new catabolic potential was independent of the presence of a Ti plasmid. The mutants were isolated after 4 weeks of incubation on minimal medium containing mannopine as the sole carbon source. They also utilized mannopinic acid, but not agropine or agropinic acid. In addition, the spontaneous mutant LM136, but not its parent strain A136, degraded many mannityl opine analogs. [14C]mannopine disappeared in the presence of LM136 cells which had been pregrown on opine or nonopine substrates. These results suggested that the catabolic system of this mutant was not subject to a stringent regulation. A clone conferring the ability to utilize mannopine on a recipient pseudomonad was selected from a genomic library from both the mutant LM136 and its parent strain. Only the LM136 clone was expressed in the parent Agrobacterium strain A136. Southern analysis showed that the genes for mannopine catabolism in the spontaneous mutants differed from the corresponding Ti plasmid-encoded genes of octopine-type or agropine-type Agrobacterium strains. Cells of LM136 utilized [14C]mannopine without generating detectable amounts of intracellular agropine. In contrast, a major fraction of the radioactivity recovered from cells of the octopine-type strain Ach5, after incubation on [14C]mannopine, was in the form of agropine.     

Resistance of Escherichia coli to penicillins. 8. Physiology of a class II ampicillin-resistant mutant

Class II ampicillin-resistant mutants of Escherichia coli are defined as having a twofold increase in penicillinase-mediated ampicillin resistance when determined by colony formation tests on plates. In this paper, one class II mutant has been compared to its parent strain. In liquid medium, the mutant was less resistant than the parent strain both in the absence and in the presence of R1 and R-factor mediating penicillinase activity. The penicillinase activity was found to be almost completely bound to the cells in the parent strain, whereas it was excreted to a great extent in the class II mutant strain. In liquid medium, resistance was well correlated to the cell-bound penicillinase activity, whereas the excreted penicillinases were also of great importance for survival on ampicillin plates. The mutant also had a changed resistance to a great number of other antibacterial drugs. The mutant was found to be more sensitive than the parent strain to osmotic shock, especially when treated with ethylenediaminetetraacetic acid or washed with sodium ions. However, the osmotic stability was restored by the presence of 1 mm Mg(2+) ions. The class II mutant was more sensitive than the parent strain to sodium cholate, and it adsorbed the phages T4 and T3-1 at a slower rate than did the parent strain. The two strains adsorbed T6 at the same rate. The class II phenotype could be gradually reversed by increasing concentrations of divalent cations. The pleiotropic changes in the phenotype are apparently unrelated to the specific targets for the antibacterial agents tested. They are secondary consequences of a cell envelope mutation. The findings indicate that the class II mutation mediates a structural change in the lipopolysaccharide of the cell envelope.     

Outer membrane protein H1 of Pseudomonas aeruginosa: involvement in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin.

It is well established that Pseudomonas aeruginosa cells grown in Mg2+-deficient medium acquire nonmutational resistance to the chelator ethylenediaminetetraacetate and to the cationic antibiotic polymyxin B; this type of resistance can be reversed by transferring the cells to Mg2+-sufficient medium for a few generations. Stable mutants resistant to polymyxin B were isolated and shown to have also gained ethylenediaminetetraacetate resistance. Both the mutants and strains grown on Mg2+-deficient medium had greatly enhanced levels of outer membrane protein H1 when compared with the wild-type strain or with revertants grown in Mg2+-sufficient medium. It was determined that in all strains and at all medium Mg2+ concentrations, the cell envelope Mg2+ concentration varied inversely with the amount of protein H1. In addition, the increase in protein H1 in the mutants was associated with an increase in resistance to another group of cationic antibiotics, the aminoglycosides, e.g., gentamicin. We propose that protein H1 acts by replacing Mg2+ at a site on the lipopolysaccharide which can otherwise be attacked by the cationic antibiotics or ethylenediaminetetraacetate.     

Elevated polysaccharide production by mutants of the fungus Aureobasidium pullulans

Abstract Two mutants of the fungus Aureobasidium pullulans ATCC 42023 were isolated that exhibited elevated polysaccharide production. Both mutants were isolated using a combination of chemical mutagenesis and resistance to growth inhibitors. It was found that both mutants elaborated higher polysaccharide levels after 7 days of growth on corn syrup or sucrose, respectively, compared to ATCC 42023. The dry weights of the mutant cells were found not to differ greatly from those of the parent cells whether corn syrup or sucrose served as the carbon source. The pullulan content of the polysaccharide synthesized by the mutants or parent cells on sucrose was consistently lower than polysaccharide synthesized on corn syrup. Using corn syrup as a carbon source, the pullulan content of the polysaccharide elaborated by the parent was higher than either mutant. The inverse was found to occur with respect to pullulan content when the strains were grown on sucrose as a carbon source.     

Role of cell wall in Saccharomyces cerevisiae mutants resistant to Hg2+.

Hg2+-resistant mutants were isolated from Saccharomyces cerevisiae. Although they were very much like the parental strains in terms of colony-forming ability, they grew faster than the parental strains in the presence of sublethal doses of Hg2+. The Hg2+-resistant mutations were dominant. They were centromere linked and were divided into two groups by means of recombination; one of the mutations, designated HGR1-1, was mapped on chromosome IV because of its linkage to the TRP1 locus. The Hg2+-resistant mutants took up Hg2+ as much as, or slightly more than, the parental strains did. The mutants and parental strains retained only about 5 and 15%, respectively, of the cell-associated Hg2+ after removal of the cell wall; therefore, the mutants had less spheroplast-associated Hg2+ than did the parental strains. These results indicate that the cell wall plays an important role in protection against Hg2+ by acting as an adsorption filter and that the mutations described confer Hg2+ resistance by increasing the Hg2+-binding capacity of the cell wall.     

Regulated exopolysaccharide production in Myxococcus xanthus

Myxococcus xanthus fibrils are cell surface-associated structures composed of roughly equal amounts of polysaccharide and protein. The level of M. xanthus polysaccharide production under different conditions in the wild type and in several mutants known to have alterations in fibril production was investigated. Wild-type exopolysaccharide increased significantly as cells entered the stationary phase of growth or upon addition of Ca2+ to growing cells, and the polysaccharide-induced cells exhibited an enhanced capacity for cell-cell agglutination. The activity of the key gluconeogenic pathway enzyme phosphoenolpyruvate carboxykinase (Pck) also increased under these conditions. Most fibril-deficient mutants failed to produce polysaccharide in a stationary-phase- or Ca2+-dependent fashion. However, regulation of Pck activity was generally unimpaired in these mutant strains. In an stk mutant, which overproduces fibrils, polysaccharide production and Pck activity were constitutively high under the conditions tested. Polysaccharide production increased in most fibril-deficient strains when an stk mutant allele was present, indicating that these fibril-deficient mutants retained the basic cellular components required for fibril polysaccharide production. In contrast to other divalent cations tested, Sr2+ effectively replaced Ca2+ in stimulating polysaccharide production, and either Ca2+ or Sr2+ was required for fruiting-body formation by wild-type cells. By using transmission electron microscopy of freeze-substituted log-phase wild-type cells, fibril material was observed as a cell surface-associated layer of uniform thickness composed of filaments with an ordered structure.     

保加利亚乳杆菌H+-ATPase缺陷型菌株的筛选

【目的】从传统乳制品中筛选具有新霉素抗性的H+-ATPase缺陷的德氏乳杆菌保加利亚亚种自发突变株,为最终开发弱后酸化的酸奶发酵剂奠定基础。【方法】利用API 50 CH细菌鉴定系统和16s rRNA基因序列分析对菌株进行鉴定。新霉素作为筛选压力,筛选具有新霉素抗性自发突变菌株,比较亲本和突变菌株的H+-ATPase活力及其代谢情况。【结果】从内蒙古地区的传统发酵酸奶中分离鉴定出一株德氏乳杆菌保加利亚亚种（Lactobacillus delbrueckii subsp. bulgaricus）,并命名为KLDS 1.9201。以此为出发菌株,筛选出两株H+-ATPase缺陷的自发突变株,分别命名为KLDS 1.9201-1、KLDS 1.9201-4,它们的H+-ATPase活力分别比亲本KLDS 1.9201降低了46%和60%。在MRS培养基中生长24 h后,KLDS 1.9201、KLDS 1.9201-1和KLDS 1.9201-4对初始葡萄糖的代谢率分别为65%、41%和31%,终产物中乳酸的浓度分别为26g/L、18g/L和15g/L,突变菌株的生物量均低于亲本。【结论】H+-ATPase活力降低的德氏乳杆菌保加利亚亚种的自发突变株具有较低的生长速率和弱产酸能力,它们可被用于制作弱后酸化的酸奶发酵剂。     

Morphology and anionic polymer content in the cell wall of a glycerol-requiring mutant ofBacillus subtilis

A glycerol-requiring mutant ofBacillus subtilis formed irregular spheres and showed disturbed septum formation, when subjected to growth limitation by the supply of glycerol. Under phosphate limitation the cells were also round and developed asymmetric septa. In magnesium-limited cultures the cells contained a thickened wall, as compared with that of the parent strain grown under the same conditions. Chemical analysis revealed the presence of teichoic acid as the major anionic polymer in the wall of the glycerol-, as well as the magnesium-limited cells of the glycerol-requiringB. subtilis mutant.Under phosphate limitation teichuronic acid was the only anionic polymer present in the wall. Thus, in this respect, there were no apparent differences between mutant organisms and the parent strain when grown under magnesium and phosphate limitation, respectively and the observed morphological deviations could not be correlated with an altered anionic polymer content of the wall.     

Differentiation between mutants of Escherichia coli K defective in oxidative phosphorylation.

Hybrid membrane particles from two mutants of Escherichia coli K12, Bv4 and K11, defective in oxidative phosphorylation, have been prepared, in which ATP-driven membrane energization is restored. A soluble factor of mutant K11 was found to have properties similar to parental crude coupling factor, ATPase (EC 3.6.1.3). Membrane particles of this mutant could not be reconstituted by parental coupling factor. Either parental coupling factor, or the soluble factor of mutant K11 could reconstitute both respiration-driven and ATP-driven energization to membrane particles of mutant Bv14 or to parental particles depleted of ATPase. Mutant Bv4 was found to be devoid of coupoing factor activity, while retaining the ability to hydrolyze ATP. Both mutants possess an ATPase with an altered binding to the membrane. Mutant K11 is impaired in respiration-driven amino acid transport, in contrast to mutant Bv4. The three major subunits of parental Escherichia coli ATPase have been isolated and antibodies have been prepared against these subunits. Antibodies against the largest subunit (alpha component) or against the intact catalytic subunits (alpha + beta components) inhibit both ATP-Pi exchange in the parent organism as well as ATP hydrolytic activity in parent and mutants. Antibodies against the two other subunits (beta or gamma components) also inhibit these two reactions, but were found to be less effective. Mutant N144, which lacks ATPase activity, shows no precipitin lines with anti-alpha, anti-beta, anti-gamma, or anti (alpha + beta) preparations. In contrast, mutants Bv4 and K11, exhibit cross-reactivity with all of the antisera.