Rhodobacter sphaeroides WS8 expresses a polypeptide that is similar to MotB of Escherichia coli.

A gene which complements a paralyzed flagellar mutant of Rhodobacter sphaeroides was sequenced. The derived protein sequence has similarity to MotB. R. sphaeroides MotB lacks the C-terminal peptidoglycan-binding motif of other MotB proteins. This divergence of sequence may reflect the unusual, unidirectional, stop-start action of the R. sphaeroides flagellar motor.     

Solubilization and purification of the MotA/MotB complex of Escherichia coli

Bacterial flagella are driven at their base by a rotary motor fueled by the membrane gradient of protons or sodium ions. The stator of the flagellar motor is formed from the membrane proteins MotA and MotB, which function together to conduct ions across the membrane and couple ion flow to rotation. An invariant aspartate residue in MotB (Asp32 in the protein of E. coli) is essential for rotation and appears to have a direct role in proton conduction. A recent study showed that changes at Asp32 in MotB cause a conformational change in the complex, as evidenced by altered patterns of protease susceptibility of MotA [Kojima, S., and Blair, D. F. (2001) Biochemistry 40 (43), 13041-13050]. It was proposed that protonation/deprotonation of Asp32 might regulate a conformational change in the stator that acts as the powerstroke to drive rotation of the rotor. Biochemical studies of the MotA/MotB complex have been hampered by the absence of a suitable assay for its integrity in detergent solution. Here, we have studied the behavior of the MotA/MotB complex in a variety of detergents, making use of the protease-susceptibility assay to monitor its integrity. Among about 25 detergents tested, a few were found to solubilize the proteins effectively while preserving certain conformational properties characteristic of an intact complex. The detergent dodecylphosphocholine, or DPC, proved especially effective. MotA/MotB complexes purified in DPC migrate with an apparent size of approximately 300 kDa in gel-filtration columns, and retain the Asp32-modulated conformational differences seen in membranes. (35)S-radiolabeling showed that MotA and MotB are present in a 2:1 ratio in the complex. Purified MotA/MotB complexes should enable in vitro study of the proton-induced conformational change and other aspects of stator function.     

New Rifampin-Resistant Mutant of Escherichia coli

A rifampin-resistant ribonucleic acid (RNA) polymerase mutant, rif(r)51, derived from a presumptive RNA synthesis mutant of Escherichia coli K-12, complements rif(r) RNA polymerase mutants isolated from other strains of E. coli K-12.     

Arrangement of core membrane segments in the MotA/MotB proton-channel complex of Escherichia coli

The stator of the bacterial flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes with stoichiometry MotA(4)MotB(2) (Kojima, S., and Blair, D. F., preceding paper in this issue). The MotA/MotB complexes conduct ions across the membrane, and couple ion flow to flagellar rotation by a mechanism that appears to involve conformational changes within the complex. MotA has four membrane-crossing segments, termed A1-A4, and MotB has one, termed B. We are studying the organization of the 18 membrane segments in the MotA(4)MotB(2) complex by using targeted disulfide cross-linking. A previous cross-linking study showed that the two B segments in the complex (one from each MotB subunit) are arranged as a symmetrical dimer of alpha-helices. Here, we extend the cross-linking study to segments A3 and A4. Single Cys residues were introduced by mutation in several consecutive positions in segments A3 and A4, and double mutants were made by pairwise combination of subsets of the Cys replacements in segments A3, A4, and B. Disulfide cross-linking of the single- and double-Cys proteins was studied in whole cells, in membranes, and in detergent solution. Several combinations of Cys residues in segments A3 and B gave a high yield of disulfide-linked MotA/MotB heterodimer upon oxidation with iodine. Positions of efficient cross-linking identify a helix face on segment A3 that is in proximity to segment(s) B. Some combinations of Cys residues in segments A4 and B also gave a significant yield of disulfide-linked heterodimer, indicating that segment A4 is also near segment(s) B. Certain combinations of Cys residues in segments A3 and A4 cross-linked to form MotA tetramers in high yield upon oxidation. The high-yield positions identify faces on A3 and A4 that are at an interface between MotA subunits. Taken together with mutational studies and patterns of amino acid conservation, the cross-linking results delineate the overall arrangement of 10 membrane segments in the MotA/MotB complex, and identify helix faces likely to line the proton channels.     

Mutant Escherichia coli Ada proteins simultaneously defective in the repair of O6-methylguanine and in gene activation.

The activated Ada protein triggers expression of DNA repair genes in Escherichia coli in response to alkylation damage. Ada also possesses two distinct suicide alkyltransferase activities, for O6-alkylguanines and for alkyl phosphotriesters in DNA. The mutant Ada3 and Ada5 transferases repair O6-methylguanine in DNA 20 and 3000 times more slowly, respectively, than the wild-type Ada protein, but both exhibit normal DNA phosphotriester repair. These same proteins also exhibit delayed and sluggish induction of the ada and alkA genes. Since the C-terminal O6-methylguanine methyltransferase domain of Ada is not implicated in the direct binding of specific DNA sequences, this part of the Ada protein is likely to play an alternative mechanistic role in gene activation, either by promoting Ada dimerization, or via direct contacts with RNA polymerase.     

Mutant of Escherichia coli deficient in osmoregulation of periplasmic oligosaccharide synthesis.

A mutant of Escherichia coli (mdoR) has been isolated which is defective in synthesis of the membrane-derived oligosaccharides (MDO) normally found in the periplasmic space. In media of high osmotic pressure this defect is suppressed and MDO levels approaching those of the wild type are produced. The mdoR mutant also fails to accumulate glycogen; however, genetic analysis showed that mdoR was not cotransducible with the known glg (glycogen) locus. A further relationship between MDO and glycogen metabolism was suggested by two observations that (i) certain glg mutants affect MDO accumulation and (ii) elevated osmotic pressure inhibits glycogen accumulation, in both wild-type and mdoR cells.     

Genetic studies of the ribosomal proteins in Escherichia coli. 3. Compositions of ribosomal proteins in various strains of Escherichia coli

Summary The comparative chromatographic investigations into the ribosomal proteins of various strains of E. coli have demonstrated that most of the strains including three strains of E. coli subsp. communior had ribosomes with the same protein compositions (C-type). The ribosomes from strain B are different from the C-type ribosomes in having the specific 30-4 (B) component in place of 30-4 (B-type), while those from strains K 12 may be distinguished from the type-C ribosomes by the presence of the specific 30-7 (K) component in place of 30-7 (K-type) or, in addition to 30-7 (K), the presence of 30-9 (W3637) in place of 30-9 (K-3637 type). Two strains, IAM 1132 and IAM 1182, have R-type ribosomes, in which at least six 50s proteins and four 30s protein components are distinct from the corresponding protein components in the C-type ribosomes.     

Structural proteins of adenovirus. Expression in Escherichia coli

The fiber proteins of adenovirus serotype 2 Ad2 and serotype 3 Ad3 and structural protein IIIa of wild type Ad2 and Ad2 ts 112 mutant were cloned and expressed in E. coli. For the expression of both fiber proteins a gene expression system based on bacteriophage T7 RNA polymerase was used. The expressed proteins constituted 1-3% of total host cell protein. Both proteins were insoluble and inclusion bodies were observed. The proteins could be purified from cellular debris by extraction with 6 M urea followed by chromatography in the presence of diminishing concentration of urea. The folding of recombinant fiber proteins was assessed by sensitivity to proteases and gel filtration. Both proteins were synthetized as trimers. Ad2 recombinant fiber has a much less compact structure than native Ad2 fiber, since on gel filtration it is excluded before the native fiber. It is also much more sensitive to chymotrypsin digestion than the native protein. Contrary to that, Ad3 recombinant fiber is much less sensitive to proteolytic cleavage and on gel filtration has the same exclusion volume as the trimeric native fiber of Ad3.     

Systematic search for zinc-binding proteins in Escherichia coli.

A systematic search for Escherichia coli proteins with the zinc-binding activity was performed using the assay of radioactive Zn(II) binding to total E. coli proteins fractionated by two methods of two-dimensional gel electrophoresis. A total of 30-40 radioactive spots were identified, of which 14 have been assigned from N-terminal sequencing. In addition to five known zinc-binding proteins, nine zinc-binding proteins were newly identified including: acetate kinase (AckA), DnaK, serine hydroxymethyltransferase (GlyA), transketolase isozymes (TktA/TktB), translation elongation factor Ts (Tsf), ribosomal proteins L2 (RplB), L13 (RplM) and one of S15 (RpsO), S16 (RpsP) or S17 (RpsQ). Together with about 20 known zinc-binding proteins, the total number of zinc-binding proteins in E. coli increased up to more than 30 species (or more than 3% of about 1000 proteins expressed under laboratory culture conditions). The specificity and affinity of zinc-binding were analysed for some of the zinc-binding proteins.     

High-affinity calcium-binding proteins in Escherichia coli

Crude extracts of Escherichia coli contain at least three heat stable proteins of Mr, 33,000, 47,000, and 60,000, which bind 45Ca2+ in buffers containing micromolar calcium and physiological salt concentrations. Fractions containing these proteins neither activated the calmodulin-dependent enzyme, NAD kinase, nor inhibited the activity of this enzyme in the presence of brain calmodulin. Radioimmunoassay of crude extracts for calmodulin indicated the presence of a calmodulin-like antigen. Crude extracts also contain proteins that interact with 2-trifluoromethyl-10H-(3'-aminopropyl)phenothiazine-Sepharose in a calcium-dependent manner, but proteins eluted from this resin did not bind calcium with high affinity.     

Evidence for interactions between MotA and MotB, torque-generating elements of the flagellar motor of Escherichia coli.

Cells that overexpress MotA (encoded on a plasmid derived from pBR322) grow slowly because of proton leakage. We have traced this defect to the coexpression of a fusion protein consisting of 60 amino acids from the N terminus of MotB and 50 amino acids specified by pBR322. Mutations within the N terminus, known to abolish function when present in full-length MotB, reversed the growth defect. Growth also was normal when MotA was coexpressed with wild-type MotB or with a series of MotB N-terminal fragments.     

Folding and assembly of oligomeric proteins in Escherichia coli.

High levels of expression of oligomeric proteins in heterologous systems are frequently associated with misfolding and accumulation of the polypeptides in inclusion bodies. This reflects aspects of the folding and assembly pathways of oligomeric proteins, which generally proceed from either folding intermediates or native-like metastable species that are not in their final conformation. Methods for optimizing the yield of correctly assembled oligomers are discussed.     

Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

NAD⁺-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD⁺ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD⁺ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.     

Isolation of Escherichia coli B Mutant Deficient in Glutathione Biosynthesis

Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 °C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles’ tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.     

Identification of the polyamine induced proteins in Escherichia coli.

The polyamine (PA)-induced proteins were identified by SDS-PAGE, and by two dimensional gel analysis in Escherichia coli strains. A large number of the PA-induced proteins were acidic. The molecular weights of the most highly induced proteins were 40 and 82 kDa proteins in the wild type and PA-auxotrophic mutant, respectively. Although a part of the PA-induced proteins were induced both in the wild type and PA auxotrophic mutant, most of them seem to be induced either in the wild type or mutant. These features may provide a foundation for evaluating the role of the PA-induced proteins relative to the physiology and environmental stress of Escherichia coli.     

Strain specificity of outer membrane proteins in Escherichia coli.

Outer membrane proteins of various strains of Escherichia coli were compared using three different systems of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The outer membranes of E. coli K-12, E. coli B, and E. coli J-5 had distinctive protein compositions. As regards proteins which interact with peptidoglycan, E. coli K-12 contained O-8 and O-9, while E. coli B possessed one protein which migrated to the position of O-9. Although E. coli J-5 possessed two such proteins, O-8' and O-9', their positions on polyacrylamide gel were different from those of O-8 and O-9. Protein O-7, which migrates slightly more slowly than O-8, was found specifically in E. coli K-12. Proteins O-10 and O-11 were found in all strains tested, although the relative amounts were different depending on the strain. Strains of E. coli K-12 and E. coli J-5 gave three major bands, O-2a, O-2b, and O-3, in the region of high molecular weight. These proteins were repressed by iron in the cultivation media. Strains of E. coli B, on the other hand, gave only O-2b and O-3. E. coli J-5 gave two other major bands in this region, but the amounts were not controlled by iron in the cultivation media.     

Mutant of Escherichia coli K-12 defective in D-glucosamine biosynthesis

A mutant was isolated from a derivative of Escherichia coli K-12 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. This mutant contained normal levels of 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10), but no detectable activity of l-glutamine:d-fructose-6-phosphate amino-transferase (EC 2.6.1.16). It required either N-acetyl-d-glucosamine or d-glucosamine for growth, and went into rapid lysis when the supply of these compounds was exhausted. In medium containing 11% sucrose, the cells were converted into spheroplasts in the absence of d-glucosamine.