A glycosyltransferase involved in biosynthesis of triglycosylated glycopeptidolipids in Mycobacterium smegmatis: impact on surface properties

The cell envelope of mycobacteria is a complex structure that plays an important role in the interactions of the cell with its environment and in the protection against the antimicrobial activity of the immune system. Glycopeptidolipids (GPLs) are species- or type species-specific glycolipids that are present at the surface of a number of mycobacteria and that are characterized by a high variability in glycosylation patterns. These GPLs possess various biological activities that depend mostly on the sugars capping the core molecule. In Mycobacterium smegmatis, the GPL core can be substituted by either two or three deoxyhexoses. In this study, we show that Gtf3 is a glycosyltransferase responsible for the synthesis of the triglycosylated GPLs. Biochemical analysis of these molecules, with a combination of mass spectrometry and chemical degradation methods, has shown that they contain three deoxyhexose moieties. The presence of the triglycosylated GPLs is associated with cell surface modifications that lead to a decrease in sliding motility as well as a modification in cellular aggregation and colony appearance on Congo red. Phylogenetic analysis indicated that Gtf3 is a member of a yet-uncharacterized glycosyltransferase family conserved among the mycobacteria.     

An electrochemical approach of the redox behavior of water insoluble ubiquinones or plastoquinones incorporated in supported phospholipid layers

Physiological mole fractions of long isoprenic chain ubiquinone (UQ[10]) and plastoquinone (PQ9) were incorporated inside a supported bilayer by vesicle fusion. The template of the bilayer was an especially designed microporous electrode that allows the direct electrochemistry of water insoluble molecules in a water environment. The artificial structure, made by self-assembly procedures, consisted of a bilayer laterally in contact with a built-in gold electrode at which direct electron transfers between the redox heads of the quinones molecules and the electrode can proceed. The mass balances of quinone and lipid in the structure were determined by radiolabeling and spectrophotometry. A dimyristoyl phosphatdylcholine stable surface concentration of 250 +/- 50 pmol x cm(-2), unaffected by the presence of the quinone, was measured in the fluid monolayer. The mole fraction of quinone was between 1 and 3 mol%, remaining unchanged when going from the vesicles to the supported layers. The lipid molecules and the quinone pool were both laterally mobile, and cyclic voltammetry was used to investigate the redox properties of UQ10 and PQ9 over a wide pH range. Below pH 12, the two electrons-two protons electrochemical process at the gold electrode appeared under kinetic control. Thus all thermodynamic deductions must be anchored in the observed reversibility of the quinone/hydroquinol anion transformation at pH > 13. Within the experimental uncertainty, the standard potentials and the pK(a)'s of the pertinent redox forms of UQ10 and PQ9 were found to be essentially identical. This differs slightly from the literature in which the constants were deduced from the studies of model quinones in mixed solvents or of isoprenic quinones without a lipidic environment.     

Potentiation of N-methyl-N'-nitro-N-nitrosoguanidine-induced O6-methylguanine-DNA-methyltransferase activity in a rat hepatoma cell line by poly (ADP-ribose) synthesis inhibitors

The O6-methylguanine-DNA-methyltransferase (transferase) activity in a rat hepatoma cell line (H4 cells) is enhanced as a response to DNA damaging agents. To study whether poly (ADP-ribosylation) is involved in this induction, the cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) that induces the transferase activity and stimulates poly (ADP-ribose) synthesis. Addition of poly (ADP-ribose) polymerase inhibitors enhanced the transferase increase induced by MNNG. The influence of the inhibitors on the transferase induction was dose and time-dependent. The results suggest that poly (ADP-ribose) is involved in the induction of this protein.     

The Escherichia coli O6-methylguanine-DNA methyltransferase does not repair promutagenic O6-methylguanine residues when present in Z-DNA

The repair of O6-methylguanine present in N-methylnitrosourea (MNU)-treated alternating polynucleotides MNU-poly(dG-dC) X poly(dG-dC) and MNU-poly(dG-me5dC) X poly(dG-me5dC] was investigated using O6-methylguanine-DNA methyltransferase purified from Escherichia coli. Both modified polynucleotides are equally good substrates for the DNA methyltransferase when they are in the B-form. The substrate properties of the MNU-treated polynucleotides do not differ from those of MNU-treated DNA. One of these modified polynucleotides, MNU-poly(dG-me5dC) X (dG-me5dC), can adopt the Z-conformation under physiological conditions. The conformational transition of the poly(dG-me5dC) X poly(dG-me5dC) from the B-form to the Z-form was monitored by the modification of its spectroscopic properties and by the specific binding of antibodies raised against Z-DNA. The O6-methylguanine residues are repaired in MNU-poly(dG-me5dC) X poly(dG-me5dC) in B-form. At variance, the conversion of this template to the Z-form completely inhibits the repair of the O6-methylguanine residues. The cooperative transition from the Z- to the B-form of MNU-poly(dG-me5dC) X poly(dG-me5dC), mediated by intercalating drugs such as ethidium bromide, restores the ability of MNU-poly(dG-me5dC) X poly(dG-me5dC) to be substrate for the transferase. These results imply that the promutagenic DNA lesion O6-methylguanine persists in Z-DNA fragments and suggest that DNA conformation modulates the extent of DNA repair and, as a result, plays an important role in determining the mutagenic potency of chemical carcinogens.     

Parallel and nonparallel behavioural evolution in response to parasitism and predation in Trinidadian guppies

Natural enemies such as predators and parasites are known to shape intraspecific variability of behaviour and personality in natural populations, yet several key questions remain: (i) What is the relative importance of predation vs. parasitism in shaping intraspecific variation of behaviour across generations? (ii) What are the contributions of genetic and plastic effects to this behavioural divergence? (iii) And to what extent are responses to predation and parasitism repeatable across independent evolutionary lineages? We addressed these questions using Trinidadian guppies (Poecilia reticulata) (i) varying in their exposure to dangerous fish predators and Gyrodactylus ectoparasites for (ii) both wild‐caught F0 and laboratory‐reared F2 individuals and coming from (iii) multiple independent evolutionary lineages (i.e. independent drainages). Several key findings emerged. First, a population's history of predation and parasitism influenced behavioural profiles, but to different extent depending on the behaviour considered (activity, shoaling or boldness). Second, we had evidence for some genetic effects of predation regime on behaviour, with differences in activity of F2 laboratory‐reared individuals, but not for parasitism, which had only plastic effects on the boldness of wild‐caught F0 individuals. Third, the two lineages showed a mixture of parallel and nonparallel responses to predation/parasitism, with parallel responses being stronger for predation than for parasitism and for activity and boldness than for shoaling. These findings suggest that different sets of behaviours provide different pay‐offs in alternative predation/parasitism environments and that parasitism has more transient effects in shaping intraspecific variation of behaviour than does predation.     

The changes in mycolic acid structures caused by hadC mutation have a dramatic effect on the virulence of Mycobacterium tuberculosis

Understanding the molecular strategies used by Mycobacterium tuberculosis to invade and persist within the host is of paramount importance to tackle the tuberculosis pandemic. Comparative genomic surveys have revealed that hadC, encoding a subunit of the HadBC dehydratase, is mutated in the avirulent M. tuberculosis H37Ra strain. We show here that mutation or deletion of hadC affects the biosynthesis of oxygenated mycolic acids, substantially reducing their production level. Additionally, it causes the loss of atypical extra‐long mycolic acids, demonstrating the involvement of HadBC in the late elongation steps of mycolic acid biosynthesis. These events have an impact on the morphotype, cording capacity and biofilm growth of the bacilli as well as on their sensitivity to agents such as rifampicin. Furthermore, deletion of hadC leads to a dramatic loss of virulence: an almost 4‐log drop of the bacterial load in the lungs and spleens of infected immunodeficient mice. Both its unique function and importance for M. tuberculosis virulence make HadBC an attractive therapeutic target for tuberculosis drug development.     

Cleavage and binding of a DNA fragment containing a single 8-oxoguanine by wild type and mutant FPG proteins.

A 34-mer oligonucleotide containing a single 7,8-dihydro-8-oxoguanine (8-OxoG) residue was used to study the enzymatic and DNA binding properties of the Fpg protein from E. coli. The highest rates of incision of the 8-OxoG containing strand by the Fpg protein were observed for duplexes where 8-OxoG was opposite C (*G/C) or T (*G/T). In contrast, the rates of incision of duplexes containing 8-OxoG opposite G (*G/G) and A (*G/A) were 5-fold and 200-fold slower. Gel retardation studies showed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where the 8-OxoG was opposite purines. KDapp values were 0.6 nM (*G/C), 1.0 nM (*G/T), 6.0 nM (*G/G) and 16.0 nM (*G/A). The Fpg protein also binds to unmodified (G/C) duplex and a KDapp of 90 nM was measured. The cleavage and binding of the (*G/C) duplex were also studied using bacterial crude lysates. Wild type E. coli crude extract incised the 8-OxoG containing strand and formed a specific retardation complex with the (*G/C) duplex. These two reactions were mediated by the Fpg protein, since they were not observed with a crude extract from a bacterial strain whose fpg gene was inactivated. Furthermore, we have studied the properties of 6 mutant Fpg proteins with Cys-->Gly mutations. The results showed that the 2 Fpg proteins with Cys-->Gly mutations outside the zinc finger sequence cleaved the 8-OxoG containing strand, formed complexes with the (*G/C) duplex and suppressed the mutator phenotype of the fpg-1 mutant. In contrast, the 4 Fpg proteins with Cys-->Gly mutations within the zinc finger motif neither cleave nor bind the (*G/C) duplex, nor do these proteins suppress the fpg-1 mutator phenotype.     

Two rotameric forms of open ring 7-methylguanine are present in alkylated polynucleotides

High performance liquid chromatography analysis of imidazole open ring 7-methylguanine, 2-6 diamino-4-hydroxy-5N-methyl-formamidopyrimidine (rom7G), showed two well-separated peaks (fI and fII) of the same magnitude. Rechromatography of each isolated component indicated that they are slowly interconverted to give a 1:1 mixture. NMR analysis demonstrated that the two species observed on reversed phase HPLC are rotational isomers. Thermodynamic measurements strongly suggested that the equilibrium can be assigned to rotation around the N-methyl formamido bond. The two species, fI and fII, separated by HPLC were identified as rotamers E and Z, respectively. The structures of fI and fII were also determined. A polynucleotide containing rom7G was obtained by alkaline treatment of poly (dGC) containing 7-methylguanine. In order to study its structure within the polynucleotide, rom7G was enzymatically excized by E.coli rom7G-DNA glycosylase. The analysis of the products released by the enzyme showed a 1:4 mixture of the two rotamers favoring the Z form (fII).     

Dopamine-stimulated cyclic AMP and binding of [3H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10^?8 M and was half-maximal at $\text{7.9±3.4·10}{}^{\mathrm{?7}}\text{M}$. The increase at 1·10^?5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10^?9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10^?5 M dopamine was $\text{2.3±0.9·10}{}^{\mathrm{?6}}\text{M}$. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value for high affinity binding sites was $\text{0.43±0.1·10}{}^{\mathrm{?7}}\text{M}$ and $\text{4.7±1.6·10}{}^{\mathrm{?7}}\text{M}$ for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was $\text{1.2±0.4·10}{}^{\mathrm{/t-7}}\text{M}$ noradrenaline, 2·10^/t-7 M epinine, $\text{4.1±1.8·10}{}^{\mathrm{/t-6}}\text{M}$ fluphenazine, $\text{8.0±1.6·10}{}^{\mathrm{/t-6}}\text{M}$ haloperidol, $\text{4.2±1.2·10}{}^{\mathrm{?6}}\text{M}$cis-flupenthixol, $\text{2.7±0.4·10}{}^{\mathrm{?5}}\text{M}$trans-flupenthixol, $\text{>1·10}{}^{\mathrm{?5}}\text{M}$ apomorphine, sulpiride, naloxone and isoproterenol.     

cDNA cloning of the rat O6-methylguanine-DNA-methyltransferase

A cDNA expression library was constructed from a rat hepatoma cell line ( H4 cells ) and introduced into an Escherichia coli strain ( BK2110 ) deficient in the repair of O6-methylguanine residues. Following three exposures to N-methyl-N'-nitro-N-nitrosoguanidine, a resistant colony harboring a plasmid named RMGMT was isolated. Extracts of BK2210 cells hosting the RMGMT plasmid expressed a O6-methylguanine-DNA-methyltransferase (transferase) activity and this protein had the same molecular weight as the transferase from H4 cells. The cDNA sequence of 763 bp contains an open reading frame of 630 bp encoding a protein of 209 amino acids with a calculated molecular weight of 22.2 kd. The rat protein shows 68% homology with the human transferase.