Selection and characterization of aerobic bacteria capable of degrading commercial mixtures of low-ethoxylated nonylphenols.

AIMS: Isolation and characterization of new bacterial strains capable of degrading nonylphenol ethoxylates (NPnEO) with a low ethoxylation degree, which are particularly recalcitrant to biodegradation. METHODS AND RESULTS: Seven aerobic bacterial strains were isolated from activated sludges derived from an Italian plant receiving NPnEO-contaminated wastewaters after enrichment with a low-ethoxylated NPnEO mixture. On the basis of 16S rDNA sequence, the strains were positioned into five genera: Ochrobactrum, Castellaniella, Variovorax, Pseudomonas and Psychrobacter. Their degradation capabilities have been evaluated on two commercial mixtures, i.e. Igepal CO-210 and Igepal CO-520, the former rich in low ethoxylated congeners and the latter containing a broader spectrum of NPnEO, and on 4-n-nonylphenol (NP). The strains degraded Igepal CO-210, Igepal CO-520 and 4-n-NP all applied at the initial concentration of 100 mg l(-1), by 35-75%, 35-90% and 15-25%, respectively, after 25 days of incubation. CONCLUSIONS: Some of the isolated strains, in particular the Pseudomonas strains BCb12/1 and BCb12/3, showed interesting degradation capabilities towards low ethoxylated NPnEO congeners maintaining high cell vitality. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased knowledge of bacteria involved in NPnEO degradation and the possibility of using the isolated strains in tailored process for a tertiary biological treatment of effluents of wastewater treatment plants.

Colony density influences invasive and filamentous growth in Saccharomyces cerevisiae.

The effect of colony density on the dimorphic switch was determined in natural strains of Saccharomyces cerevisiae. In some strains invasiveness and pseudohyphal (PH) growth were highly sensitive to colony density; moreover, strains constitutively able to invade the substrate with PH formation positively influenced the invasiveness but not the PH growth of a different strain less prone to the dimorphic switch.

A Heidenhain variant of Creutzfeldt-Jakob disease: forensic implication.

To investigate whether typical clinical, diagnostic and neuropathological findings can be identified in a patient with a postmortem diagnosis of a Heidenhain variant of Creutzfeldt-Jakob disease (CJD). We report a new case of CJD in a rare variant. A man admitted to hospital with cefalea and vision disorder. Clinical and neurological examination showed headache, vision reduction, psychomotor anxiety and progressive torpor. The patient died 4 h after admission to hospital. The autopsy findings included marked encephalic vascular congestion. Hystoneurology examination showed no macroscopic anomaly. Microscopy findings included neuronal loss, gliosis in striate area with arachnoid cells and cerebellum microspongiosis. Creutzfeldt-Jakob disease is a rare neurodegenerative human disorder. The prion hypothesis as an explanatory model is currently favoured by majority of researchers. A disease course described by Heidenhain including the leading symptoms of a visual disorder and rapid progression. This report emphasize the multidisciplinary role (forensic, neurogenetic and neurohistologic) for diagnosis and to standardize a protocol to investigate.

Biodiversity and horizontal gene transfer in culturable bacteria isolated from activated sludge enriched in nonylphenol ethoxylates.

One hundred and twenty bacterial isolates, from activated sludge of a treatment plant collecting wastes enriched in ethoxylated nonylphenols, were studied. Sixty isolates were selected on rich medium and 60 on mineral medium containing two nonylphenol ethoxylates as the sole carbon source. Analysis of biodiversity at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNA amplified by PCR from 120 isolates. The rDNA restriction analysis enabled us to cluster the isolates into 15 groups, five of which represented nearly 77% of the community. Phylogenetic analysis of five strains belonging to these main groups made it possible to assign four of them to the genera Acinetobacter, Aeromonas and Shewanella and one to the Proteus group. The analysis of plasmid content showed a high variability and suggested that horizontal gene transfer had taken place at the intraspecific, interspecific and intergeneric levels.

Identification by functional analysis of the gene encoding alpha-isopropylmalate synthase II (LEU9) in Saccharomyces cerevisiae.

The function of the open reading frame (ORF) YOR108w of Saccharomyces cerevisiae has been analysed. The deletion of this ORF from chromosome XV did not give an identifiable phenotype. A mutant in which both ORF YOR108w and LEU4 gene have been deleted proved to be leucine auxotrophic and alpha-isopropylmalate synthase (alpha-IPMS)-negative. This mutant recovered alpha-IPMS activity and a Leu(+) phenotype when transformed with a plasmid copy of YOR108w. These data and the sequence homology indicated that YOR108w is the structural gene for alpha-IPMS II, responsible for the residual alpha-IPMS activity found in a leu4Delta strain. The leu4Delta strain appeared to be very sensitive to the leucine analogue trifluoroleucine. In the absence of leucine, its growth was not much impaired in glucose but more on non-fermentable carbon sources.

Trifluoroleucine resistance as a dominant molecular marker in transformation of strains of Saccharomyces cerevisiae isolated from wine.

The resistance to 5,5,5-trifluoro-DL-leucine, encoded by the dominant allele LEU4-1, was used as a selectable marker to transform laboratory and natural Saccharomyces cerevisiae strains by the lithium acetate procedure. Results of transformation of S. cerevisiae laboratory and wine natural strains showed that trifluoroleucine resistance is a very effective selection marker and can be widely used to transform prototrophic S. cerevisiae strains. The LEU4-1 gene could also be exploited to improve wine flavour, as indicated by the higher isoamyl alcohol content of the transformants compared to the parental strains.

Disruption and phenotypic analysis of six novel genes from chromosome IV of Saccharomyces cerevisiae reveal YDL060w as an essential gene for vegetative growth.

The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN. PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids.

Trifluoroleucine resistance and regulation of alpha-isopropyl malate synthase in Saccharomyces cerevisiae.

Seven spontaneous Saccharomyces cerevisiae mutants that express dominant resistance to 5,5,5-trifluoro-DL-leucine have been characterised at the molecular level. The gene responsible for the resistance was cloned from one of the mutants (FSC2.4). Determination of its nucleotide sequence showed that it was an allele of LEU4 (LEU4-1), the gene that encodes alpha-isopropyl malate synthase I (alpha-IPM synthase I), and that the mutation involved a codon deletion localised close to the 3' end of the LEU4 ORF. Six different point mutations--four transitions and two transversions--were found in the remaining mutants. Alpha-IPM synthase activity was found to be insensitive to feedback inhibition by leucine in five of the strains. In the other two the enzyme was resistant to Zn2+-mediated inactivation by Coenzyme A, a previously postulated control mechanism in energy metabolism; as far as we know, this represents the first direct in vivo evidence for this mechanism. The seven mutations define a region, the R-region, involved in both leucine feedback inhibition and in Zn2+-mediated inactivation by CoA. Deletion experiments involving the R-region showed that it is also necessary for enzyme activity.

Biodiversity of an Acinetobacter population isolated from activated sludge.

A culturable microbial community from a sewage treatment plant collecting mainly surfactant-enriched wastes was selected on minimal medium containing two nonylphenol ethoxylates as sole carbon source. Biodiversity of the community was assessed on fifty randomly chosen isolates by a combination of molecular techniques. Isolates were first analysed by amplified 16S ribosomal DNA restriction analysis (ARDRA); most of them (75%) were assigned to the genus Acinetobacter on the basis of 16S ribosomal DNA sequencing. Random amplified polymorphic DNA (RAPD) fingerprinting and the analysis of plasmid content showed a high degree of genetic variability and suggested a marked horizontal gene transfer.

Paralogous histidine biosynthetic genes: evolutionary analysis of the Saccharomyces cerevisiae HIS6 and HIS7 genes.

The HIS6 gene from Saccharomyces cerevisiae strain YNN282 is able to complement both the S. cerevisiae his6 and the Escherichia coli hisA mutations. The cloning and the nucleotide sequence indicated that this gene encodes a putative phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxiamide isomerase (5' Pro-FAR isomerase, EC 5.3.1.16) of 261 amino acids, with a molecular weight of 29,554. The HIS6 gene product shares a significant degree of sequence similarity with the prokaryotic HisA proteins and HisF proteins, and with the C-terminal domain of the S. cerevisiae HIS7 protein (homologous to HisF), indicating that the yeast HIS6 and HIS7 genes are paralogous. Moreover, the HIS6 gene is organized into two homologous modules half the size of the entire gene, typical of all the known prokaryotic hisA and hisF genes. The structure of the yeast HIS6 gene supports the two-step evolutionary model suggested by Fani et al. (J. Mol. Evol. 1994; 38: 489-495) to explain the present-day hisA and hisF genes. According to this idea, the hisF gene originated from the duplication of an ancestral hisA gene which, in turn, was the result of an earlier gene elongation event involving an ancestral module half the size of the extant gene. Results reported in this paper also suggest that these two successive paralogous gene duplications took probably place in the early steps of molecular evolution of the histidine pathway, well before the diversification of the three domains, and that this pathway was one of the metabolic activities of the last common ancestor. The molecular evolution of the yeast HIS6 and HIS7 genes is also discussed.

Genetic and biochemical characterization of Saccharomyces cerevisiae mutants resistant to trifluoroleucine.

Eighteen mutants resistant to 5',5',5'-trifluoroleucine (TFL), a leucine analog, were isolated in Saccharomyces cerevisiae strains YNN281 and YNN282. The mutants were characterized genetically and clustered in two groups, one comprising all the dominant (TFL1) and the other one all the recessive (tfl2) mutations. Genetic and biochemical data suggested that the dominant mutations are located on the LEU4 gene, coding for alpha-isopropylmalate synthase I. These mutations resulted in accumulation of leucine as a consequence of the synthesis of an enzyme insensitive to the feedback inhibition by leucine. Leucine excretion in the TFL1 mutants appeared to be affected by the genetic background of the strain and was greatly influenced by lysine metabolism. The measurement of intra- and extracellular amino acid concentrations in prototrophic strains carrying TFL1 or tfl2 genes showed that both were leucine overproducers. Some of the TFL-resistant mutants were tested in alcoholic fermentation of grape must: analysis of the fermentation secondary metabolites showed that the major effect of the TFL-resistant strains was an increased production of isoamyl alcohol compared to that of the parental strain.

DNA fingerprinting by random amplified polymorphic DNA and restriction fragment length polymorphism is useful for yeast typing.

Random amplified polymorphic DNA (RAPD) analysis was applied to genomic DNA from nineteen yeast strains belonging to the genera Saccharomyces and Zygosaccharomyces. Results obtained with five primers indicated that this technique is a powerful tool for yeast differentiation and identification. The data were consistent with those derived from restriction fragment length polymorphism (RFLP) using two S. cerevisiae DNA probes. We conclude that RAPD fingerprinting, combined with the analysis of RFLP, can provide unambiguous type assignment in yeasts.

DNA fingerprinting of yeast strains by restriction enzyme analysis.

Restriction endonuclease analysis was used as a new method to obtain genomic DNA fingerprints in yeast. Fifteen yeast strains belonging to the genera Saccharomyces and Zygosaccharomyces were examined. Restriction fragments obtained with ApaI or KspI endonucleases were separated by SDS-PAGE and silver-stained. Analysis of the fingerprints showed that restriction enzyme digestion of genomic DNA can be successfully applied to yeast, enabling the differentiation between strains belonging to different or to the same species or genera.

Cutaneous venom of Bombina variegata pachypus (Amphibia, Anura): effects on the growth of the human HL 60 cell line.

The effects of Bombina variegata cutaneous venom (Bvv) on eukaryotic cell growth has been assessed employing the human leukaemic cell line HL 60, by liquid and agar semisolid cultures and 51Cr release assay. HL 60 cells growth is impaired by Bvv in a dose-dependent fashion in both culture systems. The arrest of proliferation requires a contact time lower than 3 min and it is not reversed by washing and culturing the cells in a Bvv-free medium. Similarly, an extremely short exposure time is needed to determine maximum 51Cr release. Neither the agar medium nor the fetal calf serum interact with Bvv effects, which, according to the above findings, must be regarded as cytolytic in nature. In both liquid and the agar-semisolid culture Bvv cytolytic activity half life is about 8 hr. The cytolytic properties of Bvv are thought to be part of the chemical defence system of amphibian skin.

A bactericidal protein in Bombina variegata pachypus skin venom.

The skin venom of the yellow bellied toad Bombina variegata pachypus has an antimicrobial activity which seems to be correlated to the presence of a 6700 mol. wt polypeptide. This polypeptide was purified by electroelution from SDS-urea-polyacrylamide gels and characterized for its antimicrobial activity. A bactericidal action was detected at concentrations with little or no cytolytic effect. The determination of the Minimal Inhibitory Concentration showed that there was activity against gram positive and gram negative bacteria and also against yeasts. The skin secretions of three other anuran species (Bufo viridis, Hyla arborea and Discoglossus pictus) were examined for the presence of antimicrobial activities. Only the Hyla arborea secretion exhibited antimicrobial properties. A small amount of a 6700 mol. wt polypeptide was detected among the Hyla secreted products.

Deontological aspects in recombinant DNA researches.

Legal problems exist in regard to patent rights for new biotechnologies. Ethical problems arise in connection with the application of the technique to patients and with the possibility to cure embryos. The potentialities of preventive medicine have affected even hiring criteria, in that individuals who present enzymic deficiencies can be considered at risk when working in contact with certain substances. For example, firms could decide not to advance certain individuals to positions of responsibility on the grounds that they present the gene of familial hypercholesterolemia. It can be anticipated that the diagnostic potentialities of the recombinant DNA technique will be applied to the evaluation of risk for the stipulation of life insurance, and that further developments, permitting to determine the genetic predisposition for most diseases, would altogether nullify the basic principles of health- and life-insurance policies. An international committee will have to discuss these issues and to formulate deontological rules to regulate both the sphere of occupation and that of insurance.

Isolation of Bacillus subtilis transformation-deficient mutants and mapping of competence genes.

We have isolated and characterized 48 Bacillus subtilis competence-deficient mutants. The mutants, obtained by nitrosoguanidine mutagenesis or by insertional mutagenesis with transposon Tn917, had a reduced transformation frequency and a wild-type transduction frequency. The com mutations were mapped by PBS1 transduction and at least four new com genes have been identified. The mutants were also characterized for their capacity to bind and take up the transforming DNA.

A study of several red cell enzyme markers in two samples of the Italian population. Report of new CA1 and PGD variant phenotypes.

Gene frequencies for 17 red cell enzymatic markers have been determined in two samples of the Italian population (Lombardy and Tuscany regions). A significant difference was found between the two samples for the AK1 and PGM1 systems (AK1*2 .028 and .044, PGM1*2 .254 and .301 in Lombardy and Tuscany respectively). Variant phenotypes, for PEPA, PEPB, CA2, PGM2, PGD and GPT markers, have been observed; some of these are due to new alleles occurring at the CA2 and PGD loci.

A low molecular weight protein with antimicrobial activity in the cutaneous 'venom' of the yellow-bellied toad (Bombina variegata pachypus).

The cutaneous 'venom' was collected from dorsal skin fragments of the yellow-bellied toad Bombina variegata pachypus by means of stimulation with noradrenaline. Light and electron microscope observations gave evidence that the 'venom' corresponds to the secretory products of both serous gland types (i.e. with small or large granules) characteristic of this genus, which had discharged their contents upon stimulation. The serous 'venom', when tested for antimicrobial activity, inhibited the growth of several bacterial strains. Heat treatment, dialysis, protease digestion and SDS-PAGE electrophoresis showed that the antimicrobial activity was thermostable and associated with a low molecular weight protein. This protein was purified and homogeneity determined by CM-cellulose chromatography and SDS-PAGE electrophoresis. The purified protein has a molecular weight of 6700, displays antibacterial properties and appears different from the antimicrobially active peptides previously isolated from the 'venom' of the toad.

Competence proteins in Bacillus subtilis com mutants.

The synthesis of nucleases and proteins specific for competence development have been studied in four different Bacillus subtilis competence-deficient mutants. The nuclease analysis showed that two DNA-binding-deficient mutants were impaired in three nuclease activities involved in binding and entry of donor DNA. The other two strains did not show any reduction in nuclease activities. Two-dimensional gel electrophoresis of the proteins, synthesized during competence development, revealed that all four mutants are lacking several competence-specific polypeptides. Our data show that these com mutations have a strong pleiotropic effect, which could be due to a block in the metabolic pathway leading to competence development.