Dimer formation of octaprenyl-diphosphate synthase (IspB) is essential for chain length determination of ubiquinone

Ubiquinone (Q), composed of a quinone core and an isoprenoid side chain, is a key component of the respiratory chain and is an important antioxidant. In Escherichia coli, the side chain of Q-8 is synthesized by octaprenyl-diphosphate synthase, which is encoded by an essential gene, ispB. To determine how IspB regulates the length of the isoprenoid, we constructed 15 ispB mutants and expressed them in E. coli and Saccharomyces cerevisiae. The Y38A and R321V mutants produced Q-6 and Q-7, and the Y38A/R321V double mutant produced Q-5 and Q-6, indicating that these residues are involved in the determination of chain length. E. coli cells (ispB::cat) harboring an Arg-321 mutant were temperature-sensitive for growth, which indicates that Arg-321 is important for thermostability of IspB. Intriguingly, E. coli cells harboring wild-type ispB and the A79Y mutant produced mainly Q-6, although the activity of the enzyme with the A79Y mutation was completely abolished. When a heterodimer of His-tagged wild-type IspB and glutathione S-transferase-tagged IspB(A79Y) was formed, the enzyme produced a shorter length isoprenoid. These results indicate that although the A79Y mutant is functionally inactive, it can regulate activity upon forming a heterodimer with wild-type IspB, and this dimer formation is important for the determination of the isoprenoid chain length.     

Quinones of phototrophic purple bacteria

Abstract The quinone composition of the recognized species of the phototrophic purple nonsulfur bacteria, the Ectothiorhodospiraceae, and some Chromatiaceae species has been determined. Altogether more than 50 strains of 33 species have been investigated. Some of the purple nonsulfur bacteria have Q-10 as sole quinone component, while others have Q-10, Q-9, or Q-8, respectively, together with menaquinones of the same isoprenoid chain length as the major components. Rhodoquinone is present in Rhodospirillum rubrum and Rhodospirillum photometricum . The Ectothiorhodospira species have either Q-8 and MK-8, like the Chromatiaceae species, or Q-7 and MK-7 as the major components.     

Characterization of recombinant Desulfovibrio gigas ferredoxin.

Dg ferredoxin gene was cloned using the polymerase chain reaction (PCR), inserted into vector pT7-7, and overexpressed in Escherichia coli (E. coli) grown in aerobic media. The recombinant protein is a dimer and contains a [3Fe-4S] cluster per monomer. EPR and (1)H NMR data of recombinant and wild-type protein are compared.     

Cloning and nucleotide sequence of the fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli.

We report the cloning and nucleotide sequence of the gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli. Malonyl transacylase has been overexpressed 155-fold compared to a wild-type strain. Overexpression of this enzyme alters the fatty acid composition of a wild-type E. coli strain; increased amounts of cis-vaccenate are incorporated into the membrane phospholipids.     

Menaquinone biosynthesis in Escherichia coli: identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate as a novel intermediate and re-evaluation of MenD activity

Menaquinone is an electron carrier in the respiratory chain of Escherichia coli during anaerobic growth. Its biosynthesis involves (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) as an intermediate, which is believed to be derived from isochorismate and 2-ketoglutarate by one of the biosynthetic enzymes-MenD. However, we found that the genuine MenD product is 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid (SEPHCHC), rather than SHCHC. This is supported by the following findings: (i) isochorismate consumption and SHCHC formation are not synchronized in the enzymic reaction, (ii) the rate of SHCHC formation is independent of the enzyme concentration, (iii) SHCHC is not formed in weakly acidic or neutral solutions in which the isochorismate substrate is readily consumed by MenD, and (iv) the MenD turnover product, formed under conditions disabling SHCHC formation, possesses spectroscopic characteristics consistent with the structure of SEPHCHC and spontaneously undergoes 2,5-elimination to form SHCHC and pyruvate in weakly basic solutions. Two properties of the intermediate, ultraviolet transparency and chemical instability, provide a rationale for the fact that SHCHC has been consistently mistaken as the MenD product. In accordance with these findings, MenD was rediscovered to be a highly efficient enzyme with a high second-order rate constant and should be renamed SEPHCHC synthase. Intriguingly, the enzymatic activity responsible for conversion of SEPHCHC into SHCHC appears not to associate with any of the known enzymes in menaquinone biosynthesis but is present in the crude extract of E. coli K12, suggesting that a genuine SHCHC synthase remains to be identified to fully elucidate the ubiquitous biosynthetic pathway.     

Quinone profiling of bacterial communities in natural and synthetic sewage activated sludge for enhanced phosphate removal

Respiratory quinones were used as biomarkers to study bacterial community structures in activated sludge reactors used for enhanced biological phosphate removal (EBPR). We compared the quinone profiles of EBPR sludges and standard sludges, of natural sewage and synthetic sewage, and of plant scale and laboratory scale systems. Ubiquinone (Q) and menaquinone (MK) components were detected in all sludges tested at molar MK/Q ratios of 0.455 to 0.981. The differences in MK/Q ratios were much larger when we compared different wastewater sludges (i.e., raw sewage and synthetic sewage) than when we compared sludges from the EBPR and standard processes or plant scale and laboratory scale systems. In all sludges tested a Q with eight isoprene units (Q-8) was the most abundant quinone. In the MK fraction, either tetrahydrogenated MK-8 or MK-7 was the predominant type, and there was also a significant proportion of MK-6 to MK-8 in most cases. A numerical cluster analysis of the profiles showed that the sludges tested fell into two major clusters; one included all raw sewage sludges, and the other consisted of all synthetic sewage sludges, independent of the operational mode and scale of the reactors and the phosphate accumulation. These data suggested that Q-8-containing species belonging to the class Proteobacteria (i.e., species belonging to the beta subclass) were the major constituents of the bacterial populations in the EBPR sludge, as well as in standard activated sludge. Members of the class Actinobacteria (gram-positive bacteria with high DNA G+C contents) were the second most abundant group in both types of sludge. The bacterial community structures in activated sludge processes may be affected more by the nature of the influent wastewater than by the introduction of an anaerobic stage into the process or by the scale of the reactors.     

Crystal structure of the Tyr45Trp mutant of ribonuclease T1 in a complex with 2'-adenylic acid

The recombinant Tyr45Trp mutant of Lys25-ribonuclease T1 was overexpressed and purified from an Escherichia coli strain. The mutant enzyme, which shows reduced activity towards GpA and increased activity towards pGpC, pApC and pUpC compared with wild-type RNase T1, was co-crystallized with 2'-adenylic acid by microdialysis. The space group is P212121 with unit cell dimenions a = 4.932(2), b = 4.661(2), c = 4.092(1) nm. The crystal structure was solved using the coordinates of the isomorphous complex of wild-type RNase T1 with 2'-AMP. The refinement was based on Fhkl of 7726 reflexions with Fo greater than or equal to 1 sigma (Fo) in the resolution range of 2.0-0.19 nm and converged with an R factor of 0.179. The adenosine of 2'-AMP is not bound to the guanosine binding site, as could be expected from the mutation of Tyr45Trp, but is stacked on the Gly74 carbonyl group and the His92 imidazole group which form a subsite for substrate binding, as already observed in the wild-type 2'-AMP complex. The point mutation of Tyr45Trp does not perturb the backbone conformation and the Trp-indole side chain is in a comparable position to the phenolic Tyr45 of the wild-type enzyme.     

An unusual twin-his arrangement in the pore of ammonia channels is essential for substrate conductance

Amt proteins constitute a class of ubiquitous integral membrane proteins that mediate movement of ammonium across cell membranes. They are homotrimers, in which each subunit contains a narrow pore through which substrate transport occurs. Two conserved histidine residues in the pore have been proposed to be necessary for ammonia conductance. By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance. Crystal structures of variants confirm that substitution of the histidine residues does not affect AmtB structure. In a subgroup of Amt proteins, found only in fungi, one of the histidines is replaced by glutamate. The equivalent substitution in E. coli AmtB is partially active, and the structure of this variant suggests that the glutamate side chain can make similar interactions to those made by histidine.     

A harmonic structure of the genetic code

In this paper is presented a new, very harmonic structure of the genetic code (GC) within a system of "4 x 5" (and/or of "5 x 4") of amino acids (AAs) in two variants. In first variant, the five rows within the system start with one polar charged amino acid (AA) each, making first column, consisting from five polar charged AAs (D, R, K, H, E). Five polar non-charged AAs (N, P, Y, W, Q) follow, then five non-polar AAs as last column (A, L, F, V, I) and, finally, five polar or non-polar AAs, in a combination, as first to last column (A as non-polar; S, T as polar, and G, P as ambivalent AAs). A second variant is subsequent to this one-"4 x 5" system with five nitrogen AAs (K, R, P, H, W), five oxygen (D, E, Y, S, T), five solely carbon (A, L, F, V, I) and five "combined" AAs (G with hydrogen as side chain; C and M with carbon and sulfur; N and Q with carbon, oxygen and nitrogen). A strict balance of atom and nucleon number as well as molecule mass follows the classification in both system variants.     

Cell division in Escherichia coli minB mutants

In Escherichia coli minB mutants, cell division can take place at the cell poles as well as non-polarly in the cell. We have examined growth, division patterns, and nucleoid distribution in individual cells of a minC point mutant and a minB deletion mutant, and compared them to the corresponding wild-type strain and an intR1 strain in which the chromosome is over-replicated. The main findings were as follows. In the minB mutants, polar and non-polar divisions appeared to occur independently of each other. Furthermore, the timing of cell division in the cell cycle was found to be severely affected. In addition, nucleoid conformation and distribution were considerably disturbed. The results obtained call for a re-evaluation of the role of the MinB system in the E. coli cell cycle, and of the concept that limiting quanta of cell division factors are regularly produced during the cell cycle.     

Kinetic behavior of phosphotriesterase and its non-covalent complexes with polyelectrolyte in systems with polar and non-polar organic solvents

Soluble phosphotriesterase from E. coli DH5 together with E. coli DH5 cells with the phosphotriesterase activity were co-immobilized into poly(vinyl alcohol) (PVA) cryogel and studied in water/organic systems with polar and non-polar organic solvents. The phosphotriesterase activity was competitively inhibited by polar organic solvents. The inhibition constant correlated with the dielectric constant () of the solvent. The rate of the enzyme-catalyzed reaction in biphasic non-polar solvent/water systems was independent of water/organic ratio and the hydrophobicity of the solvent. Formation of the non-covalent complexes with polyelectrolytes was suggested to enhance the resistance of the phosphotriesterase towards inactivation by organic solvents in their homogeneous mixtures with water.     

A conserved glycine residue of trimeric autotransporter domains plays a key role in Yersinia adhesin A autotransport

The Yersinia adhesin A (YadA) is a trimeric autotransporter adhesin of enteric yersiniae. It consists of three major domains: a head mediating adherence to host cells, a stalk involved in serum resistance, and an anchor that forms a membrane pore and is responsible for the autotransport function. The anchor contains a glycine residue, nearly invariant throughout trimeric autotransporter adhesins, that faces the pore lumen. To address the role of this glycine, we replaced it with polar amino acids of increasing side chain size and expressed wild-type and mutant YadA in Escherichia coli. The mutations did not impair the YadA-mediated adhesion to collagen and to host cells or the host cell cytokine production, but they decreased the expression levels and stability of YadA trimers with increasing side chain size. Likewise, autoagglutination and resistance to serum were decreased in these mutants. We found that the periplasmic protease DegP is involved in the degradation of YadA and that in an E. coli degP deletion strain, mutant versions of YadA were expressed almost to wild-type levels. We conclude that the conserved glycine residue affects both the export and the stability of YadA and consequently some of its putative functions in pathogenesis.     

Isolation of a naphthaquinone with partly hydrogenated side chain from Corynebacterium diphtheriae

1. Corynebacterium diphtheriae contains relatively large amounts (6.6mumoles/g. dry wt.) of a naphthaquinone whose ultraviolet-absorption spectrum is that of a typical menaquinone (vitamin K(2)), the E(1%) (1 cm.) value corresponding with that of MK-8, but on reversed-phase paper chromatograms it runs with MK-9. 2. In the presence of Adams catalyst hydrogen uptake is 2 atoms/mol. less than that calculated for MK-8. 3. Hydrogenated samples of the Corynebacterium quinone and the hydrogenation product of authentic MK-8 ran together on reversed-phase chromatograms. 4. Infrared-absorption spectra indicated close relationship with the menaquinone series, and nuclear-magnetic-resonance measurements show that one, non-terminal, double bond of the side chain has been saturated. 5. The compound is thus designated MK-8(2H), indicating a menaquinone with eight isoprene units but only seven double bonds in the side chain.     

Kinetics and substrate specificities of desulfo-glucosinolate sulfotransferases in Arabidopsis thaliana

Sulfotransferases (SOTs) (EC 2.8.2.-) catalyze the transfer of a sulfate group from the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a hydroxyl group of different substrates. In Arabidopsis thaliana , three SOTs were identified to catalyze the last step of glucosinolate (Gl) core structure biosynthesis called AtSOT16, 17 and 18. These enzymes from Arabidopsis ecotype C24 were overexpressed in Escherichia coli and purified by affinity chromatography. Recombinant proteins were used to determine substrate specificities to investigate whether each of the three desulfo (ds)-Gl SOTs might influence the Gl pattern of Arabidopsis differently. After optimization of the enzyme assay, it was possible to measure in vivo substrates with non-radioactive PAPS by HPLC analysis of the product. In vitro enzyme assays revealed a preference of AtSOT16 for the indolic ds-Gl indol-3-yl-methyl, AtSOT17 showed an increased specific activity with increasing chain length of ds-Gl derived from methionine and AtSOT18 preferred the long-chain ds-Gl, 7-methylthioheptyl and 8-methylthiooctyl, derived from methionine. In planta ds-Gl exist side by side; therefore, initial results from one substrate measurements were verified using a defined mixture of ds-Gl and ds-Gl/Gl leaf extracts from Arabidopsis ecotype C24. These studies confirmed the one substrate measurements. To compare SOTs from different Arabidopsis ecotypes, additionally, AtSOT18* from ecotype Col-0 was overexpressed in E. coli and purified. The recombinant protein was used for in vitro measurements and revealed a different enzymatical behavior compared with AtSOT18 from C24. In conclusion, there are differences in the substrate specificities between the three ds-Gl AtSOT proteins within ecotype C24 and differences among ds-Gl AtSOT18 proteins from different ecotypes.     

An AP site can protect against the mutagenic potential of 8-oxoG when present within a tandem clustered site in E. coli

Ionizing radiation induces clustered DNA damaged sites, defined as two or more lesions formed within one or two helical turns of the DNA through passage of a single radiation track. It is now established that clustered DNA damage sites are found in cells and present a challenge to the repair machinery of the cell but to date, most studies have investigated the effects of bi-stranded lesions. A subset of clustered DNA damaged sites exist in which two or more lesions are present in tandem on the same DNA strand. In this study synthetic oligonucleotides containing an AP site 1, 3 or 5 bases 5' or 3' to 8-oxo-7,8-dihydroguanine (8-oxoG) on the same DNA strand were synthesized as a model of a tandem clustered damaged sites. It was found that 8-oxoG retards the incision of the AP site by exonuclease III (Xth) and formamidopyrimidine DNA glycosylase (Fpg). In addition the rejoining of the AP site by xrs5 nuclear extracts is impaired by the presence of 8-oxoG. The mutation frequency arising from 8-oxoG within a tandem clustered site was determined in both wild type and mutant E. coli backgrounds. In wild-type, nth, fpg and mutY null E. coli, the mutation frequency is slightly elevated when an AP site is in tandem to 8-oxoG, compared with when 8-oxoG is present as a single lesion. Interestingly, in the double mutant mutY/fpg null E. coli, the mutation frequency of 8-oxoG is reduced when an AP site is present in tandem compared with when 8-oxoG is present as a single lesion. This study demonstrates that tandem lesions can present a challenge to the repair machinery of the cell.     

Role of ceramide structure and its microenvironment on the conformational order of model stratum corneum lipids mixtures: an approach by FTIR spectroscopy

The aim of this study is to investigate the influence of ceramide head group architecture and free fatty acid (another main class of stratum corneum lipids) or protein (keratin), on the lamellar organization of the ceramide auto-associated in model films mimicking lipid organization within the stratum corneum. FTIR spectroscopy is a powerful technique for investigating the structure of such systems. This technique has already been used to characterize phase transitions of the SC and of related model systems. As temperature is known to modify the conformational order of lipids, we used it as a variable parameter to monitor the differences in the conformational stability of ceramides. Our study included four ceramides: ceramide 2, 3, 5 and 6 which differ by their head group architecture. Two kinds of lipid-lipid interactions were studied: non-polar and polar. We noted some structural factors which participated to the organizational behavior: insaturation of alkyl chain, alpha-hydroxyl on fatty acid moiety and sphingosine or phytosphingosine head group. There is a direct interaction of palmitic acid on alkyl chains organization and a weak interaction with polar head group in presence of keratin, both provoking a destabilization of the ceramidic orthorhombic organization.     

Membrane phospholipid synthesis in Escherichia coli: alteration by glycerol and physiological consequences in a pss mutant

Escherichia coli mutants harboring the pss-1 allele (coding for a temperature-sensitive phosphatidylserine synthase) are temperature sensitive for growth and synthesize less phosphatidylethanolamine at higher temperatures, giving rise to abnormal membrane phospholipid compositions. To obtain information concerning the determinant for the phospholipid polar headgroup composition and the lethal factor in the defective membranes, we have examined the effect of increased supply of sn-glycerol 3-phosphate on the phospholipid synthesis and the growth ability of a pss-1 mutant. For this purpose, a pair of E. coli K-12 derivatives isogenic except for the pss-1 allele was constructed from strain BB26-36 to harbor the mutations related to glycerol metabolism (glpD3, glpR2, glpKi, and phoA8). Pulse- and uniform-labeling of phospholipids with 32P at 42 degrees C in a synthetic medium with (0.2%) or without glycerol showed that glycerol further lowered the temperature-sensitive formation of phosphatidylethanolamine, removed the phosphatidate and CDP-diacylglycerol accumulated in the absence of glycerol, and resulted in an increase in cardiolipin content in the pss-1 mutant. The phospholipid synthesis and contents in the pss+ strain were not significantly affected by glycerol. Glycerol in the medium markedly enhanced the growth defect of the pss-1 mutant, which was remediable by sucrose. The results indicate that the intracellular pool of sn-glycerol 3-phosphate is the limiting factor for acidic phospholipid synthesis in the pss-1 mutant, and cardiolipin unusually accumulated is injurious to the functional E. coli membranes. Possible determinants for the phospholipid composition of the wild-type E. coli cells are also discussed on the basis of the present observations.     

Isolation and analysis of a mutant of Escherichia coli hyper-resistant to near-ultraviolet light plus 8-methoxypsoralen

A mutant of Escherichia coli K12 was isolated which shows enhanced resistance towards near-ultraviolet (NUV) light plus 8-methoxypsoralen (MPS) compared with its wild-type parent strain. The PUVA (NUV + MPS)-resistant strain remains as sensitive for far-ultraviolet (FUV) light as its parent strain. A recA- derivative of this mutant strain was as sensitive to PUVA as its reca- parental strain. A polyacrylamide gel electrophoresis study of total cell lysates from the mutant bacteria showed that a protein of approximately 55 kd was synthesised in higher concentrations compared with its synthesis in the wild-type parent strain. Furthermore, synthesis of this protein was reduced in the recA- derivative of the mutant strain suggesting that the recA gene product might be acting as a regulator of the synthesis of the 55-kd protein. It is suggested that in E. coli damage to DNA by PUVA can be repaired by a specific RecA LexA-inducible repair system and the repair efficiency is enhanced if the 55-kd protein is present in concentrations higher than that synthesised by the wild-type parent E. coli.     

Hydrophobic thickness, lipid surface area and polar region hydration in monounsaturated diacylphosphatidylcholine bilayers: SANS study of effects of cholesterol and beta-sitosterol in unilamellar vesicles

The influence of a mammalian sterol cholesterol and a plant sterol beta-sitosterol on the structural parameters and hydration of bilayers in unilamellar vesicles made of monounsaturated diacylphosphatidylcholines (diCn:1PC, n=14-22 is the even number of acyl chain carbons) was studied at 30 degrees C using small-angle neutron scattering (SANS). Recently published advanced model of lipid bilayer as a three-strip structure was used with a triangular shape of polar head group probability distribution (Kucerka et al., Models to analyze small-angle neutron scattering from unilamellar lipid vesicles, Physical Review E 69 (2004) Art. No. 051903). It was found that 33 mol% of both sterols increased the thickness of diCn:1PC bilayers with n=18-22 similarly. beta-sitosterol increased the thickness of diC14:1PC and diC16:1PC bilayers a little more than cholesterol. Both sterols increased the surface area per unit cell by cca 12 A(2) and the number of water molecules located in the head group region by cca 4 molecules, irrespective to the acyl chain length of diCn:1PC. The structural difference in the side chain between cholesterol and beta-sitosterol plays a negligible role in influencing the structural parameters of bilayers studied.