Specificity of L,D-transpeptidases from gram-positive bacteria producing different peptidoglycan chemotypes

We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the L,D-transpeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. The lone functionally characterized member of this new family of active site cysteine peptidases, Ldt(fm) from Enterococcus faecium, was previously shown to bypass the D,D-transpeptidase activity of the classical penicillin-binding proteins leading to high level cross-resistance to glycopeptide and beta-lactam antibiotics. Ldt(fm) homologues from Bacillus subtilis (Ldt(Bs)) and E. faecalis (Ldt(fs)) were found here to cross-link their cognate disaccharide-peptide subunits containing meso-diaminopimelic acid (mesoDAP(3)) and L-Lys(3)-L-Ala-L-Ala at the third position of the stem peptide, respectively, instead of L-Lys(3)-d-iAsn in E. faecium. Ldt(fs) differed from Ldt(fm) and Ldt(Bs) by its capacity to hydrolyze the L-Lys(3)-D-Ala(4) bond of tetrapeptide (L,D-carboxypeptidase activity) and pentapeptide (L,D-endopeptidase activity) stems, in addition to the common cross-linking activity. The three enzymes were specific for their cognate acyl acceptors in the cross-linking reaction. In contrast to Ldt(fs), which was also specific for its cognate acyl donor, Ldt(fm) tolerated substitution of L-Lys(3)-D-iAsn by L-Lys(3)-L-Ala-L-Ala. Likewise, Ldt(Bs) tolerated substitution of mesoDAP(3) by L-Lys(3)-D-iAsn and L-Lys(3)-L-Ala-L-Ala in the acyl donor. Thus, diversification of the structure of peptidoglycan precursors associated with speciation has led to a parallel evolution of the substrate specificity of the L,D-transpeptidases affecting mainly the recognition of the acyl acceptor. Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving L,D-transpeptidases.     

Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the beta-lactam imipenem

The beta-lactam antibiotics mimic the D-alanyl(4)-D-alanine(5) extremity of peptidoglycan precursors and act as "suicide" substrates of the DD-transpeptidases that catalyze the last cross-linking step of peptidoglycan synthesis. We have previously shown that bypass of the dd-transpeptidases by the LD-transpeptidase of Enterococcus faecium (Ldt(fm)) leads to high level resistance to ampicillin. Ldt(fm) is specific for the L-lysyl(3)-D-alanine(4) bond of peptidoglycan precursors containing a tetrapeptide stem lacking D-alanine(5). This specificity was proposed to account for resistance, because the substrate of Ldt(fm) does not mimic beta-lactams in contrast to the D-alanyl(4)-D-alanine(5) extremity of pentapeptide stems used by the DD-transpeptidases. Here, we unexpectedly show that imipenem, a beta-lactam of the carbapenem class, totally inhibited Ldt(fm) at a low drug concentration that was sufficient to inhibit growth of the bacteria. Peptidoglycan cross-linking was also inhibited, indicating that Ldt(fm) is the in vivo target of imipenem. Stoichiometric and covalent modification of Ldt(fm) by imipenem was detected by mass spectrometry. The modification was mapped into the trypsin fragment of Ldt(fm) containing the catalytic Cys residue, and the Cys to Ala substitution prevented imipenem binding. The mass increment matched the mass of imipenem, indicating that inactivation of Ldt(fm) is likely to involve rupture of the beta-lactam ring and acylation of the catalytic Cys residue. Thus, the spectrum of activity of beta-lactams is not restricted to transpeptidases of the DD-specificity, as previously thought. Combination therapy with imipenem and ampicillin could therefore be active against E. faecium strains having the dual capacity to manufacture peptidoglycan with transpeptidases of the LD- and DD-specificities.     

Crystal structure of a novel beta-lactam-insensitive peptidoglycan transpeptidase

During the final stages of cell-wall synthesis in bacteria, penicillin-binding proteins (PBPs) catalyse the cross-linking of peptide chains from adjacent glycan strands of nascent peptidoglycan. We have recently shown that this step can be bypassed by an L,D-transpeptidase, which confers high-level beta-lactam-resistance in Enterococcus faecium. The resistance bypass leads to replacement of D-Ala4-->D-Asx-L-Lys3 cross-links generated by the PBPs by L-Lys3-->D-Asx-L-Lys3 cross-links generated by the L,D-transpeptidase. As the first structure of a member of this new transpeptidase family, we have determined the crystal structure of a fragment of the L,D-transpeptidase from E.faecium (Ldt(fm217)) at 2.4A resolution. Ldt(fm217) consists of two domains, the N-terminal domain, a new mixed alpha-beta fold, and the ErfK_YbiS_YhnG C-terminal domain, a representative of the mainly beta class of protein structures. Residue Cys442 of the C-terminal domain has been proposed to be the catalytic residue implicated in the cleavage of the L-Lys-D-Ala peptide bond. Surface analysis of Ldt(fm217) reveals that residue Cys442 is localized in a buried pocket and is accessible by two paths on different sides of the protein. We propose that the two paths to the catalytic residue Cys442 are the binding sites for the acceptor and donor substrates of the L,D-transpeptidase.     

Novel mechanism of resistance to glycopeptide antibiotics in Enterococcus faecium

Glycopeptides and beta-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-D-Ala(4)-D-Ala(5) extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and D,D-transpeptidase activities of the penicillin-binding proteins (PBPs). The beta-lactams are structural analogues of D-Ala(4)-D-Ala(5) and act as suicide substrates of the D,D-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described beta-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldt(fm)) can lead to high level resistance to glycopeptides and beta-lactams. Cross-resistance was selected by glycopeptides alone or serially by beta-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of D-Ala(5), thereby providing the substrate of Ldt(fm). Complete elimination of D-Ala(5), a residue essential for glycopeptide binding, was possible because Ldt(fm) uses the energy of the L-Lys(3)-D-Ala(4) peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala(4)-D-Ala(5) bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate.     

A novel peptidoglycan cross-linking enzyme for a beta-lactam-resistant transpeptidation pathway

The beta-lactam antibiotics remain the most commonly used to treat severe infections. Because of structural similarity between the beta-lactam ring and the d-alanyl(4)-d-alanine(5) extremity of bacterial cell wall precursors, the drugs act as suicide substrates of the dd-transpeptidases that catalyze the last cross-linking step of cell wall assembly. Here, we show that this mechanism of action can be defeated by a novel type of transpeptidase identified for the first time by reverse genetics in abeta-lactam-resistant mutant of Enterococcus faecium. The enzyme, Ldt(fm), catalyzes in vitro the cross-linking of peptidoglycan subunits in a beta-lactam-insensitive ld-transpeptidation reaction. The specificity of Ldt(fm) for the l-lysyl(3)-d-alanine(4) peptide bond of tetrapeptide donors accounts for resistance because the substrate does not mimic beta-lactams in contrast to d-alanyl(4)-d-alanine(5) in the pentapeptide donors required for dd-transpeptidation. Ldt(fm) homologues are encountered sporadically among taxonomically distant bacteria, indicating that ld-transpeptidase-mediated resistance may emerge in various pathogens.     

Inactivation kinetics of a new target of beta-lactam antibiotics

Peptidoglycan is predominantly cross-linked by serine DD-transpeptidases in most bacterial species. The enzymes are the essential targets of β-lactam antibiotics. However, unrelated cysteine LD-transpeptidases have been recently recognized as a predominant mode of peptidoglycan cross-linking in Mycobacterium tuberculosis and as a bypass mechanism conferring resistance to all β-lactams, except carbapenems such as imipenem, in Enterococcus faecium. Investigation of the mechanism of inhibition of this new β-lactam target showed that acylation of the E. faecium enzyme (Ldt(fm)) by imipenem is irreversible. Using fluorescence kinetics, an original approach was developed to independently determine the catalytic constants for imipenem binding (k(1) = 0.061 μM(-1) min(-1)) and acylation (k(inact) = 4.5 min(-1)). The binding step was limiting at the minimal drug concentration required for bacterial growth inhibition. The Michaelis complex was committed to acylation because its dissociation was negligible. The emergence of imipenem resistance involved substitutions in Ldt(fm) that reduced the rate of formation of the non-covalent complex but only marginally affected the efficiency of the acylation step. The methods described in this study will facilitate development of new carbapenems active on extensively resistant M. tuberculosis.     

The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly contains cross-links generated by L,D-transpeptidation

Our understanding of the mechanisms used by Mycobacterium tuberculosis to persist in a "dormant" state is essential to the development of therapies effective in sterilizing tissues. Gene expression profiling in model systems has revealed a complex adaptive response thought to endow M. tuberculosis with the capacity to survive several months of combinatorial antibiotic treatment. We show here that this adaptive response may involve remodeling of the peptidoglycan network by substitution of 4-->3 cross-links generated by the D,D-transpeptidase activity of penicillin-binding proteins by 3-->3 cross-links generated by a transpeptidase of L,D specificity. A candidate gene, previously shown to be upregulated upon nutrient starvation, was found to encode an L,D-transpeptidase active in the formation of 3-->3 cross-links. The enzyme, Ldt(Mt1), was inactivated by carbapenems, a class of beta-lactam antibiotics that are poorly hydrolyzed by the M. tuberculosis beta-lactamases. Ldt(Mt1) and carbapenems may therefore represent a target and a drug family relevant to the eradication of persistent M. tuberculosis.     

Differences between the complexes formed by monomeric fibrin with fragment D and dimer D

The paper is concerned with studies in formation of monomeric fibrin (fm) complexes with fragment D (D) of fibrinogen and dimer D (DD) of stabilized fibrin. The complexes are shown to be essentially different. The fm-D complexes are unstable, their composition is a function of D concentration in the mixture, the ultimate molar D/fm ratio is equal to 3. The fm-DD complexes are quite stable, their composition is constant: the molar DD/fm ratio is equal to 1. In mixtures containing fm, DD and different amounts of D complexes of different composition are formed but the total number of D-units in them approaches 3. A model is suggested showing interaction of fm molecules in protofibril formation with allowance for the retention of binding centres which provide the lateral link between protofibrils.     

Localization of cholinergic neurons in the cat lower brain stem

The localization of cholinergic neurons in the cat lower brain stem was determined immunocytochemically with a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. ChAT-positive neurons were observed in four major cell groups: cranial nerve motor and special visceromotor neurons: parasympathetic preganglionic visceromotor neurons; neurons located in the ponto-mesencephalic tegmentum including area X (or pedunculopontine tegmental nucleus), nucleus laterodorsalis tegmenti (Ldt) of Castaldi, and peri-locus coeruleus alpha (peri-alpha); and neurons located in nucleus reticularis magnocellularis (Mc) and adjacent nucleus reticularis gigantocellularis (Gc) of the medulla.     

Changes in conduction velocity, median frequency, and root mean square-amplitude of the electromyogram during 25% maximal voluntary contraction of the triceps brachii muscle, to limit of endurance

The objective of the present study was to investigate whether isometric contraction of the right triceps brachii muscle, of maximal duration and at 25% of the maximal voluntary contraction (MVC), would reduce mean fibre conduction velocity (CV) for the active motor units (MU). In addition to the cross-correlation of surface electromyograms (EMG) for CV determination, median frequency (fm) and root-mean-square amplitude (rms-amplitude) were calculated. The initial 5 min of the recovery of the three parameters was also investigated. The MVC were performed before and after the sustained contraction. Seven males-six in their twenties and one aged 43-participated in the investigation. Mean CV for the unfatigued muscle was 4.5 m.s-1, SD 0.38. On average, CV decreased less than 10% during the sustained contraction (P less than 0.05). The fm decreased almost linearly (46%) during the endurance time, while three quarters of the 250% increase in rms-amplitude took place during the last 50% of the contraction (P less than 0.001, both parameters). The MVC was reduced by 39% immediately after exhaustion (P less than 0.05). During the 1st min of recovery the rms-amplitude decreased by 50%, and the fm increased from 54% to 82% of the initial value (both P less than 0.05). No measurable simultaneous CV restitution occurred. A parallel 15% increase in fm and CV took place during the last 4 min of recovery (both P less than 0.001), while the amplitude remained constant. Since mean CV was essentially unchanged during the last 50% of the endurance time where large changes in fm and rms-amplitude occurred, factors supplementary to CV probably caused the striking changes in fatigue EMG, notably-MU recruitment, synchronization of MU activity, and lowering of MU firing frequencies. Nevertheless, during the last 4 min of recovery the entire increase in fm could be accounted for by the simultaneous increase in CV.     

Torsional rigidities of weakly strained DNAs

Measurements on unstrained linear and weakly strained large (> or =340 bp) circular DNAs yield torsional rigidities in the range C = 170-230 fJ fm. However, larger values, in the range C = 270-420 fJ fm, are typically obtained from measurements on sufficiently small (< or =247 bp) circular DNAs, and values in the range C = 300-450 fJ fm are obtained from experiments on linear DNAs under tension. A new method is proposed to estimate torsional rigidities of weakly supercoiled circular DNAs. Monte Carlo simulations of the supercoiling free energies of solution DNAs, and also of the structures of surface-confined supercoiled plasmids, were performed using different trial values of C. The results are compared with experimental measurements of the twist energy parameter, E(T), that governs the supercoiling free energy, and also with atomic force microscopy images of surface-confined plasmids. The results clearly demonstrate that C-values in the range 170-230 fJ fm are compatible with experimental observations, whereas values in the range C > or = 269 fJ fm, are incompatible with those same measurements. These results strongly suggest that the secondary structure of DNA is altered by either sufficient coherent bending strain or sufficient tension so as to enhance its torsional rigidity.     

Effect of physico-chemical properties of polyacrylamide gel media on the growth of Escherichia coli colonies]

We studied the growth of Escherichia coli LE-392 colonies on polyacrylamide gels (PAAG) depending on the physico-chemical properties of the latter, i.e. polymer concentration in the gel, swelling degree, bound water content (fm), spin-lattice relaxation and spin-spin relaxation times of water molecule protons, and modulus of elasticity (G0). S- or R-type colonies formed depending on gel properties; the diametral growth rate of S colonies was 3 times less compared with that on the control agar medium (Tryptose broth). The procedure is proposed for preparation of PAAG which rules out syneresis. Functional relations between the polymer concentrations in uniformly swelling gels and concentrations of copolymers in the reaction mixture, fm and G0 were revealed. The fm and G0 parameters can be used for controlling the quality of PAAG.     

Motility evaluation of bull spermatozoa by photon correlation spectroscopy.

Quasi-elastic light scattering techniques are employed to evaluate motility of bovine spermatozoa. The electric field correlation function CE(k, tau) has two components CE(k, tau) = alphafm+(1-alpha)fd, where fm is the correlation function due to motile cells, fd is due to dead cells and alpha is the fraction of motile cells. A function which is a linear combination of the experimentally measured fd and an empirically determined function fm is fit to the CE(k, tau) data. In this way, alpha and the approximate analytic form of fm are determined. The distribution of swimming speeds P(v) is derived from fm using an inverse Fourier sine transform procedure. Results for the time dependence of motility of bull sperm in Hank's balanced salt solution are presented.     

MCF-7; a human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors.

We have identified receptors for glucocorticoids, progestins, and androgens in a human breast tumor cell line (MCF-7) known to have estrogen receptor. Sucrose density gradients show that MCF-7 cytosol contains approximately 100 fm/mg protein estradiol (E2-3H) receptor, more than 300 fm/mg protein progesterone receptor (measured with R5020-3H), about 40 fm/mg protein 5alpha-dihydrotestosterone (5alpha-DHT-3H) receptor, and 800 fm/mg glucocorticoid receptor (measured with dexamethasone-3H). Dissociation constants obtained by Scatchard analyses were approximately 0.6 x 10(-10)M (E2), 1 x 10(-9)M (R5020), 2.8 x 10(-10)M (5alpha-DHT) and 8 x 10(-9)M (dexamethasone). No cross competition was found for estrogen receptor, but progestins competed for androgen and glucocorticoid binding. The androgen, but not the glucocorticoid, partially competed for R5020 binding to progesterone receptor. This first demonstration of 4 classes of steroid receptors in human breast cancer means that MCF-7 may be an excellent in vitro model for studying the mechanism of tumor response to endocrine therapy as well as the complex relationships between binding and biological actions of these hormones.     

Interactions of allelochemicals with dietary constituents: effects on deterrency

Abstract. That variation in the water and nutrient content of plant tissues affects allelochemical toxicity to insects is well established. However, little is known about how these dietary constituents influence allelochemical deterrency. In vertebrates, deterrency of particular allelochemicals increases with dietary water, and decreases with an increase in dietary nutrients. We determined if these findings were relevant to phytophagous insects through an experimental design that allowed us to vary independently the content of water (70–90% fresh mass, fm, with nutrient level at 10% fm) and nutrients (10–30% fm with water level at 70% or 80% fm) in an artificial diet through use of alphacel, a non-nutritive cellulose fibre. We examined the effect of these dietary manipulations on allelochemical deterrency by comparing larval feeding responses of two noctuid species, the oligophagous Anticarsia gemmatalis Hübner and the polyphagous Spodoptera frugiperda (J. E. Smith), to a control diet and an allelochemical-treated diet in two consecutive, no-choice tests. These tests were restricted to 3 min to minimize post-ingestive influences. From an initial twelve compounds tested, all but two were excluded for the following reasons: (i) failure to elicit an intermediate level of deterrency at <1% fm (i.e. albizziin, amygdalin, hordenine, ouabain, pipecolic acid, salicin, sinigrin and umbelliferone); (ii) an apparently rapid toxic effect (nicotine hydrogen tartrate); and (iii) adsorption to alphacel (quinine hydrochloride), which may have reduced deterrency. The deterrency of caffeine and linamarin increased with dietary water but was unaffected by nutrient content for both species. The similar results for an alkaloid and a cyanogenic glycoside, with two species differing considerably in feeding habits, suggest that dietary water is likely to influence the defensive efficacy of a broader range of deterrent allelochemicals to a variety of plant-feeding insects.     

Gradual Drought Under Field Conditions Influences the Glutathione Metabolism,Redox Balance and Energy Supply in Spring Wheat

Glutathione (GSH) metabolism, redox balance and energy supply in spring wheat (Triticum aestivum L.) during gradual drought stress under field conditions were investigated. Although levels of total and reduced GSH were decreased, the ratio of GSH/GSSG (glutathione disulfide) was markedly increased by drought. Levels of GSH biosynthetic precursors, cysteine (Cys) and -glutamylcysteine (-GC), and the activities of their biosynthetic enzymes, -glutamylcysteine synthetase (-GCS) and glutathione synthetase (GSHS) were also significantly increased in stressed plants. Glutathione reductase (GR) activity, which is responsible for the conversion of GSSG to GSH, was also increased under this field stress. However, two other important enzymes in GSH metabolism, glutathione peroxidase (GP) and glutathione S-transferase (GST), showed decreased activity in the droughted plants. These results suggest that the higher ratio of GSH/GSSG, the rate of GSH biosynthesis and the capacity of its redox cycling rather than GSH accumulation might be essential for drought resistance of plants. Activities of the two key Calvin-cycle enzymes possessing exposed sulfhydryl groups, NADP⁺-dependent glyceraldehydes-3-phosphate dehydrogenase (G3PD) and fructose-1,6-bisphosphatase (FBPase) were not affected by drought stress, whereas, activity of the key enzyme in the pentose-phosphate pathway (PPP), 6-phosphogluconate dehydrogenase (6-PGD), increased in the droughted plants. The ratios of NADPH/NADP⁺, NADH/NAD⁺ and ATP/ADP increased in the droughted plants, indicating that an up-regulation of the reduced redox state and the energy supply in the plant cells might be an important physiological strategy for plants responding to drought stress. A simple correlation between the high ratio of GSH/GSSG, the rate of GSH biosynthesis and the redox cycle and the high reduction states of redox status in the plant cells was also observed under field drought.     

A variant enterococcal surface protein Esp(fm) in Enterococcus faecium; distribution among food,commensal, medical,and environmental isolates

Enterococci are increasingly important causes of nosocomial disease. Also, they are associated with food and have a history of use as dairy starter and probiotic cultures. An enterococcal surface protein Esp(fs) is involved the virulence and biofilm-forming capacity of Enterococcus faecalis and recently we demonstrated the presence of a homologue Esp(fm) in E. faecium. Here we describe the complete structure of Esp(fm) and demonstrate that its distribution in E. faecium correlates with disease associated strains from a range of pathological sites.     

Influence of shade timing on an Equisetum arvense L. population

Equisetum arvense L. is a perennial pteridophyte that grows in open sites. In Tokyo, the plant has photosynthetic shoots from late March to November. However, in some populations, these shoots are lost before summer because of shading by taller plants. To investigate the contribution of shoots that remain on the plant for a certain duration, in terms of the maintenance of the E.arvense population, tubers were cultivated under different light conditions and the dry weight of growth, photosynthetic rates and respiration rates were measured. Individual growth was simulated on the basis of matter production and its partitioning. Biomass at the start of the next growing season (the initial size) was seriously decreased by shading before July. However, shading after July had little effect on the initial size of the next season plants. Thus, E.arvense can maintain its population if its shoots are retained until summer.     

The Lower-Middle Pleistocene succession of the Coastal Tuscany (Central Italy): new stratigraphic and palaeoecological data based on the ostracod fauna

The Lower-early Middle Pleistocene succession of the Coastal Tuscany is known to comprise three marine cycles: (I) a Santernian-Emilian cycle; (II) a Sicilian (“small Gephyrocapsa” Zone) cycle; (III) a third cycle, referred through stratigraphic and palaeoethnological arguments to the late Sicilian-early Middle Pleistocene, including the fluvial-transitional San Marco fm and the shoreface to backshore sandy-arenitic deposits of the correlatable Bibbona and Casa Saracino formations, outcropping in the Bibbona (Lower Cecina Valley) and Rosignano areas respectively. Conversely to the older cycles the third one has been poorly studied and its chronology and depositional history have remained somewhat uncertain. With the aim to fill this gap of knowledge the sedimentary record exposed in the Rosignano and Bibbona areas was the object of new on field investigations and microfaunal content (chiefly ostracods) analyses. Furthermore, this has represented a good opportunity to enhance our knowledge of the Pleistocene Mediterranean ostracods. The main results achieved are in synthesis the followings. (1) Stratigraphic and palaeoenvironmental significance of ostracods from the first cycle is consistent with literature data. Unexpectedly the recovered assemblages comprise both warm-temperate species (e.g. Cytherelloidea beckmanni Barbeito-Gonzales, Propontocypris solida Ruggieri, Verrucocythereis bulbuspinata (Uliczny), which suggest a relatively warm climate phase, and an yet undescribed species of Ruggieria, a genus previously thought to be represented in the Italian Lower Pleistocene only by Ruggieria nuda Moyes. (2) In agreement with previous studies, sediments of the San Marco fm in the Rosignano area are referable to a floodplain-coastal lagoonal setting. Divergently from literature data, in the Bibbona area the unit exhibits vuggy carbonate glaebules and rizhoconcrections and yields very rare fresh-brackish water ostracods and marine microfaunas regarded as reworked. Despite interpretation of these sediments still poses many problems, we speculate that they represent marine deposits reworked in a poorly drained continental-transitional environment, which experienced pedogenic alteration. Furthermore, the common occurrence of the ostracode Aurila puncticruciata Ruggieri seems to support the supposition that reworked deposits included marine Sicilian sediments completely eroded and presumably correlatable to the Fabbriche fm. (3) Lithological-sedimentological features and absence of autochthonous macro-microfossils indicate that the Casa Saracino fm and most of the Bibbona unit accumulated in a backshore environment dominated by aeolian deposition. Only locally the latter unit includes shallow marine deposits with fairly rich ostracod faunas, which confidently indicate an age not younger than the Sicilian sensu Ruggieri and Sprovieri [Riv. Mineraria Siciliana 151/153 (1975) 1]. Thus, it seems possible that the Coastal Tuscany succession includes two marine cycles, which developed within the Sicilian.     

Efficient decontamination of zearalenone, the mycotoxin of cereal pathogen, by transgenic yeasts through the expression of a synthetic lactonohydrolase gene

Zearalenone (ZEN), an estrogenic mycotoxin produced by several Fusarium species, is converted to a non-estrogenic product by a detoxifying enzyme of Clonostachys rosea. Previously, we investigated whether recombinant Saccharomyces cerevisiae carrying this detoxification gene, zhd101, can remove 2 g ml^–1 of ZEN in a liquid culture. Although the transgenic yeasts eliminated most of the ZEN, they also converted a significant amount to a poor substrate, -zearalenol, which remained in the medium. In this study, we synthesized a codon-optimized zhd101 gene and investigated whether the transgenic yeast strain can overcome the problem of insufficient detoxification of ZEN. Importantly, within 48 h of incubation at 28°C or 8 h of incubation at 37°C, the transgenic yeasts completely eliminated 2 g ml^–1 of ZEN in the medium without accumulating even a trace amount of -zearalenol. The result suggests that incomplete ZEN detoxification attributed to the action of an endogenous yeast -reductase can be overcome by simply increasing the expression of the detoxifying gene.