An extracellular lipopolysaccharide-phospholipid-protein complex produced by Escherichia coli grown under lysinelimiting conditions

Lysine limitation during growth of the lysine-requiring mutant of Escherichia coli 12408 resulted in the excretion of a complex containing 60% of lipopolysaccharide, 26% of extractable phospholipid and 11% of protein. The complex was obtained from the culture filtrate in yields of about 0·5g./l. by precipitation with chloroform or gel filtration; some purification steps are described. The greater part of the phospholipid consisted of phosphatidylethanolamine, which contained four main fatty acids; two monoenoic acids and a cyclopropane acid were esterified mainly in the β-position, and a saturated acid was located mainly in the γ-position. The protein component was relatively insoluble and contained an excess of acidic over basic amino acids and little cystine. The lipopolysaccharide resembled in composition the intracellular lipopolysaccharides from rough strains of E. coli. Both protein and lipopolysaccharide constituents of the complex were serologically active; the complex was less toxic than the purified lipopolysaccharide. In the electron microscope the complex showed a mixture of particles of various sizes and shapes. Rods and hollow spheroids (diameter 12–200mμ) were most common and resembled the particles previously found surrounding cells actively excreting the complex. The chloroform-precipitated material showed a tubular lamellar structure. Soluble lipopolysaccharide prepared from the complex also consisted of hollow spheres and rods.     

COMPARATIVE PEPTIDE MAPPING AND ISOELECTRIC FOCUSING OF ISOLATED SUBUNITS FROM CHICK EMBRYO BRAIN TUBULIN

The α- and β-subunits of chick embryo brain tubulin have been isolated under denaturing conditions and compared with respect to their molecular weight, amino acid composition, tryptic peptide maps, amide content and isoelectric focusing properties. An 8 M-Urea-containing polyacrylamide gel system with varying acrylamide concentrations was used for calculation of the retardation coefficients (K_R) of the tubulin subunits. A molecular weight of 53,000 was estimated for each subunit by comparison to K_R values for standard proteins. Amide contents of approx 41% of the carboxyl groups of α-tubulin and 48% of the carboxyl groups of β-tubulin were calculated using the average PI value, the pK_intrinsic for the ionizable side chains of the amino acids and the amino acid composition of each subunit. Comparative peptide maps of trypsin digested α- and β-tubulin demonstrated 16 peptides unique to each subunit and 23 peptides which comigrate. Both subunits give rise to multiple species on electrofocusing gels. The average isoelectric points for the α- and β-subunits are 5.4 and 5.2, respectively.     

X-ray diffraction studies of bacterial pili

A sample of bacterial pili was prepared from Escherichia coli. An X-ray diffraction pattern was obtained from an oriented wet gel specimen in 0.01 m-phosphate buffer (pH 7.0) packed in a capillary tube. Sixteen independent spots were observed with the spacing of the outermost being 4.2 Å. Analysis of the diffraction pattern shows that the arrangement of subunits in pili rods is strictly simple-helical with 3.14₅ units being present in one turn of the helix and the axial rise per unit being 8.09_o Å.     

Purification and characterization of a surface-associated carotenoid-binding complex from the photosynthetic prokaryote,Prochlorothrix hollandica

We have identified a water-soluble surface-associated complex from Prochlorothrix hollandica, composed of two polypeptides of 56 and 58 kilodaltons (kDa), zeaxanthin, and lipopolysaccharide. The complex was purified by preparative isoelectric focusing (pI=3.0). The outer membrane lipopolysaccharide co-purified with the complex. Immunocytochemisty employing a polyclonal antibody to the apoproteins exclusively labeled the cell surface. Both zeaxanthin and the protein accumulated under high light intensities, thus we propose that the complex may play a role in photoprotection.     

The pKa Values of Acidic and Basic Residues Buried at the Same Internal Location in a Protein Are Governed by Different Factors

The pK_a values of internal ionizable groups are usually very different from the normal pK_a values of ionizable groups in water. To examine the molecular determinants of pK_a values of internal groups, we compared the properties of Lys, Asp, and Glu at internal position 38 in staphylococcal nuclease. Lys38 titrates with a normal or elevated pK_a, whereas Asp38 and Glu38 titrate with elevated pK_a values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys38 side chain makes an ion pair with Glu122, whereas in the structure of the L38E variant, the buried carboxyl group of Glu38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously, we showed that the pK_a of Lys38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pK_a values of Asp38 and Glu38 are perturbed significantly owing to an imbalance between favorable polar interactions and unfavorable contributions from dehydration and from Coulomb interactions with surface carboxylic groups. Their ionization is also coupled to subtle structural reorganization. These results illustrate the complex interplay between local polarity, Coulomb interactions, and structural reorganization as determinants of pK_a values of internal groups in proteins. This study suggests that improvements to computational methods for pK_a calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize.     

Quantification of bacterial adherence on different textile fabrics

This research studied the adherent behaviour of gram-negative Escherichia coli on different weft knitted textile fabrics made of cotton, polyester filaments and polyester (staple)-cotton blended yarn. We compared the bacterial adherence of 18-h-old E. coli cells on all the three types of fabrics under the same experimental conditions. The maximum adherence was found in cotton, followed by the polyester blend; the least adherence was in polyester fabrics. Scanning electron micrographs showed that surface morphology of fabrics also plays an important role during adherence. Cotton fabric, with a rough surface, attracted more bacterial cells compared to the smooth polyester surface. Comparing the FTIR spectra of different fabrics and E. coli it was found that both cotton and E. coli have abundant free hydroxyl groups that may interact strongly with each other and with other hydrophilic groups such as carboxyl, phosphate, and amides. This may be one of the reasons for the greater adherence on cotton as compared to hydrophobic polyester fabric. Finally, the effect of bacterial adherence on loss of strength in different fabrics was analysed. It was found that the maximum decrease in strength occurred in cotton fabrics and the least in polyester fabrics. The present study suggests a procedure for quantifying bacterial adherence on different textile fabrics. This technique can be used with different bacterial strains and varieties of fabrics for quantifying the adherent bacterial cells, and would be of great use in developing and comparing different antimicrobial finished products of the textile industry.     

A retrospective: Use of Escherichia coli as a vehicle to study phospholipid synthesis and function

Although the study of individual phospholipids and their synthesis began in the 1920s first in plants and then mammals, it was not until the early 1960s that Eugene Kennedy using Escherichia coli initiated studies of bacterial phospholipid metabolism. With the base of information already available from studies of mammalian tissue, the basic blueprint of phospholipid biosynthesis in E. coli was worked out by the late 1960s. In 1970s and 1980s most of the enzymes responsible for phospholipid biosynthesis were purified and many of the genes encoding these enzymes were identified. By the late 1990s conditional and null mutants were available along with clones of the genes for every step of phospholipid biosynthesis. Most of these genes had been sequenced before the complete E. coli genome sequence was available. Strains of E. coli were developed in which phospholipid composition could be changed in a systematic manner while maintaining cell viability. Null mutants, strains in which phospholipid metabolism was artificially regulated, and strains synthesizing foreign lipids not found in E. coli have been used to this day to define specific roles for individual phospholipid. This review will trace the findings that have led to the development of E. coli as an excellent model system to study mechanisms underlying the synthesis and function of phospholipids that are widely applicable to other prokaryotic and eukaryotic systems. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Purification and Properties of γγ-Enolase from Pig Brain

Isoelectric focusing revealed three enolase isoforms in pig brain, which were designated as αα- (pI = 6.5), αγ- (pI = 5.6), and γγ-enolase (pI = 5.2). The pI of purified γγ-enolase was also 5.2. The γγ-enolase isoform of enolase was purified from pig brain by a purification protocol involving heating to 55°C for 3 min, acetone precipitation, ammonium sulfate precipitation (40%–80%), DEAE Sephadex ion-exchange chromatography (pH 6.2), and Sephadex G200 gel filtration. The final specific activity was 82 units/mg protein. As with other vertebrate enolases, γγ-enolase from pig proved to be a dimer with a native mass of 85 kDa and a subunit mass of 45 kDa. The pH optimum for the reaction in the glycolytic direction is 7.2. The K _m values for 2-PGA, PEP, and Mg²⁺ were determined to be 0.05, 0.25, and 0.50 mM, respectively, similar to K _m values of other vertebrate enolases. The amino acid composition of pig γγ-enolase, as determined by amino acid analysis, shows strong similarity to the compositions of γγ-enolases from rat, human, and mouse, as determined from their amino acid sequences. Despite the differences seen with some residues, and considering the ways that the compositions were obtained, it is assumed that pig γγ-enolase is more similar than the composition data would indicate. Moreover, it is likely that the sequences of pig γγ-enolase and the other γγ-enolases are almost identical. Li⁺ proved to be a noncompetitive inhibitor with either 2-PGA or Mg²⁺ as the variable substrate. This enolase crystallized in the monoclinic space group P2, or P2₁. An R _symm <5% was obtained for data between 50 and 3.65 Å, but was a disappointing 30% for data between 3.65 and 3.10 Å, indicating crystal disorder.     

Nano silver entrapped in phospholipids membrane: Synthesis,characteristics and antibacterial kinetics

Abstract

The antimicrobial property of stabilized silver nanoparticles (AgNPs) with phospholipid membrane was investigated on both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacterial strains. The influence of phospholipid concentrations on antibacterial kinetics actions of AgNPs was studied with two different methodologies in order to understand the bactericidal and bacteriostatic effects. The bacterial inactivation of synthesized AgNPs fitted well to the Chick-Watson model with a high regression coefficient, R² > 0.91. The antibacterial properties of AgNPs depend on the particle size, stabilizer and lecithin concentrations. Only the stabilized AgNPs that have the K_lec/Ag values of 1 and 2 presented the inhabitation zone, while unstabilized AgNPs agglomerated quickly, settled on the wells and did not diffuse in agar. In addition, the specific coefficient of lethality depends on the lecithin concentration. An increase in lecithin concentration caused multilayer creation on the AgNPs' surface and reduced the release of AgNPs which led to low bacterial killing rate.     

Ion-exchange properties of the cell walls isolated from suspension-cultured plant cells

Ion-exchange capacity of the cell walls isolated from suspension-cultured Panax japonicus, Polyscias filicifolia and Dioscorea deltoidea cells was analyzed at pH 2.8–12 and constant ionic strength (100 mM). The cell walls of all cultures contain three types of ion-exchange groups: primary amino groups (pK _a < 3), carboxyl groups of polygalacturonic acid (pK _a 3.71), and carboxyl groups of hydroxycinnamic acids (pK _a 7.62). Amount of primary amino groups ranges from 500 (D. deltoidea) to 710 (P. japonicus) µmol/g cell wall dry weight, carboxyl groups with pK _a 3.71—from 570 (D. deltoidea) to 670 (P. filicifolia), carboxyl groups with pK _a 7.62—from 270 (P. filicifolia) to 370 (P. japonicus) µmol/g cell wall dry weight. The comparison of the data obtained by elemental and functional analyses demonstrated that the cell walls of all cultures are characterized by high content of pectins (~40% by weight) and structural proteins (~17–30% by weight), but do not contain phenolic OH–groups, which presumably signifies the absence of lignin in them.     

Purification and crystallization of a complex between human interferon γ receptor (extracellular domain) and human interferon γ

X-ray diffraction quality crystals have been obtained from a complex between interferon γ and the extracellular domain of its high-affinity cell surface receptor. The crystals were obtained from interferon γ/interferon γ receptor complexes purified by size exclusion chromatography. Diffraction quality crystals required analyzing these complex samples by isoelectric focusing gels to select purified complex fractions devoid of unbound interferon γ. These studies used interferon γ receptor engineered with an eight amino acid N-terminal deletion to eliminate heterogeneity generated due to proteolytic cleavage. In addition, the receptor was expressed in an E. coli secretion cell line which eliminated the need to refold the protein. Hexagonal crystals were grown from 1.6 M ammonium phosphate solutions and belong to a spacegroup of P6₅22 with unit cell dimensions a = 145.9 Å and c = 180.3 Å. These crystals diffract to at least 2.9 Å resolution when exposed to synchrotron radiation. SDS PAGE analysis of the crystals demonstrated that both interferon γ and the receptor were present. Analysis of the x-ray diffraction data revealed that the crystals contain complexes with a stoichiometry of 2:1 receptor: ligand within the crystallographic asymmetric unit and consist of approximately 55% solvent. © 1996 Wiley-Liss, Inc.     

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA^Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure–function understanding in prokaryotic tRNA^Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA^Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA^Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed.     

Molecular recognition of lipopolysaccharide by the lantibiotic nisin

Nisin is a lanthionine antimicrobial effective against diverse Gram-positive bacteria and is used as a food preservative worldwide. Its action is mediated by pyrophosphate recognition of the bacterial cell wall receptors lipid II and undecaprenyl pyrophosphate. Nisin/receptor complexes disrupt cytoplasmic membranes, inhibit cell wall synthesis and dysregulate bacterial cell division. Gram-negative bacteria are much more tolerant to antimicrobials including nisin. In contrast to Gram-positives, Gram-negative bacteria possess an outer membrane, the major constituent of which is lipopolysaccharide (LPS). This contains surface exposed phosphate and pyrophosphate groups and hence can be targeted by nisin. Here we describe the impact of LPS on membrane stability in response to nisin and the molecular interactions occurring between nisin and membrane-embedded LPS from different Gram-negative bacteria. Dye release from liposomes shows enhanced susceptibility to nisin in the presence of LPS, particularly rough LPS chemotypes that lack an O-antigen whereas LPS from microorganisms sharing similar ecological niches with antimicrobial producers provides only modest enhancement. Increased susceptibility was observed with LPS from pathogenic Klebsiella pneumoniae compared to LPS from enteropathogenic Salmonella enterica and gut commensal Escherichia coli. LPS from Brucella melitensis, an intra-cellular pathogen which is adapted to invade professional and non-professional phagocytes, appears to be refractory to nisin. Molecular complex formation between nisin and LPS was studied by solid state MAS NMR and revealed complex formation between nisin and LPS from most organisms investigated except B. melitensis. LPS/nisin complex formation was confirmed in outer membrane extracts from E. coli.     

Glucose 6-phosphate dehydrogenase isozymes of maize leaves : some comparative properties

Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP⁺ to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP⁺. The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves.

Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, K_m of NADP⁺ being 2.5-fold higher for pI 4.3 form. Free ATP (K_m = 0.64 millimolar) and ADP (K_m = 1.13 millimolar) act as competitive inhibitors with respect to NADP⁺ in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition.

     

A Novel Bacterial Enzyme with d-Glucuronyl C5-epimerase Activity

Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of d-glucuronic acid to its C5-epimer l-iduronic acid, which is essential for the function of heparan sulfate. Although l-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic d-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with d-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human d-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial d-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans.     

The stacking of chloroplast thylakoids: Quantitative analysis of the balance of forces between thylakoid membranes of chloroplasts,and the role of divalent cations

An analysis is made of the van der Waals dispersion attractive forces and electrostatic repulsive forces between the grana thylakoid membranes of chloroplasts. These forces are determined for negatively charged surfaces with a pK_a value of 4.7 for a bulk pH of 7.0 with a range of mono- and divalent cation concentrations and intermembrane spacing in the range 10 to 80 Å. For equilibrium under dark conditions, it is concluded that either there is extensive electrostatic binding of divalent cations (Mg²⁺) to the negatively charged membrane groups (phospholipid, sulfolipid, and protein carboxyl), or a redistribution of these groups between stacked and unstacked regions must be invoked.     

Spinach leaf phosphoenolpyruvate carboxylase: purification, properties, and kinetic studies

Spinach leaf phosphoenolpyruvate carboxylase has been purified to homogeneity using salt fractionatjon, chromatography, and immunologie procedures to remove contaminating ribulose diphosphate carboxylase. From gel filtration and isoelectric focusing, the molecular weight (~560,000) and isoelectric point (pI = 4.9) are indistinguishable from those of ribulose diphosphate carboxylase. The subunit molecular weight of phosphoenolpyruvate carboxylase (130,000) suggests that the native enzyme is a tetramer.Kinetic studies using Mg²⁺ or Mn²⁺ as the activator indicate that the divalent cation lowers the K_m of the substrate phosphoenolpyruvate by an order of magnitude and conversely, that the presence of the substrate similarly lowers the K_m of the metal ion, suggesting an enzyme-metal-substrate bridge complex. Three analogs of phosphoenolpyruvate, lphospholactate, d-phospholactate, and phosphoglycolate are potent competitive inhibitors. The inhibitor constant (K_i) of l-phospholactate (2 μm) is 49-fold lower with Mn²⁺ as the activator than with Mg²⁺. An analysis of the competitive inhibition by portions of the l-phospholactate molecule (i.e., l-lactate, methyl phosphate, and phosphite) indicates this 49-fold lowering is due to increased interaction of the phosphoryl group and, to a lesser extent, of the carboxyl and C-O-P bridge oxygen of l-phospholactate with the enzyme metal complex. The results provide indirect evidence for phosphoryl coordination by the enzyme-bound divalent cation.     

Mutual Relationship between Antibiotics and Resting Spores of Bacillus subtilis: Binding of Cyclic Polypeptide and Aminoglycoside Antibiotics to Spores and Their Inhibitory Effect on Outgrowth and Vegetative Growth

Not only cyclic polypeptide antibiotics such as polymyxin B, colistin and gramicidin S but also aminoglycoside antibiotics such as streptomycin, kanamycin, gentamicin and kanamycin derivatives combined with the resting spores of Bacillus subtilis and inhibited outgrowth or vegetative growth after germination. All the antibiotics other than gramicidin S were released from the resting spores and their inhibitory action was reversed by the addition of Ca²⁺ and Fe³⁺. As the above antibiotics have free amino (or guanidine) groups in common, it was assumed that such groups play an important role in binding of the antibiotics to the resting spores. Moreover, it was shown that protamine and poly-l-lysine were also bound to the resting spores and were released from them by Ca²⁺. On the other hand, free carboxyl groups had been demonstrated in the outermost surface of the resting spores in a previous study. Thus, we assume that the mode of binding of the antibiotics to the resting spores may be due to the formation of reinforced ionic bonds between amino (or guanidine) groups in the antibiotics and carboxyl groups on the spore surface.     

Reversible envelope effects during and after killing of Escherichia coli W by a highly-purified rabbit polymorphonuclear leukocyte fraction

The effects of a highly-purified, potently bactericidal fraction from rabbit polymorphonuclear leukocytes on the envelope of Escherichia coli (W) have been examined. This leukocyte fraction has equally enriched bactericidal, permeability-increasing and phospholipase A₂ activities, and is essentially devoid of lysozyme, myeloperoxidase and protease activities (Weiss, J., Franson, R.C., Beckerdite, S., Schmeidler, K. and Elsbach, P. (1975) J. Clin. Invest. 55, 33–42). Rapid killing of E. coli by this fraction is accompanied by two almost immediate alterations in the bacterial envelope: (1) a discrete increase in envelope permeability (measured by inhibition of bacterial leucine incorporation by normally impermeant actinomycin D), and, (2) hydrolysis of ¹⁴C-labeled fatty acid-prelabeled E. coli phospholipids. Both envelope effects are promptly reversed during further incubation at 37 °C, but not at 0 °C, with 40 mM Mg²⁺. Reversal is also produced by Ca²⁺ (40 mM) and trypsin (200 μg/ml), but 200 mM K⁺ causes only partial recovery and Na⁺ and hyperosmolar sucrose are ineffective. Upon addition of Mg²⁺, phospholipid degradation ceases abruptly and the labeled products of hydrolysis (free fatty acids and lysocompounds) disappear with a corresponding reaccumulation of radioactive diacylphosphatides. The time course of resynthesis of phospholipids coincides with that of restoration of the permeability barrier. Higher concentrations of the leukocyte fraction and prolonged incubation increase both the extent of phospholipid degradation and the time required for reversal of both envelope effects. These findings suggest that both the initiation of the increased permeability and its reversal are linked to respectively the breakdown and resynthesis of major E. coli membrane phospholipids, and thus depend on the fact that the biochemical apparatus of E. coli remains capable of biosynthesis despite loss of viability.Treatment of E. coli, exposed to the leukocyte fraction, with albumin results in extracellular sequestration of the products of hydrolysis and also restores the permeability barrier to actinomycin D, suggesting that the accumulation of lytic products of lipid hydrolysis within the bacterial envelope, rather than the loss of phospholipids per se, causes increased permeability.