Anaerobic biodegradation of pentachlorophenol by a methanogenic consortium

An anaerobic consortium degrading pentachlorophenol (PCP) by methanogenic fermentation was enriched from PCP-contaminated soils. In a semi-continuous reactor, PCP biodegradation was unstable and necessitated periodic additions of unacclimated anaerobic sludge waste to restore the activity. In continuous-flow reactors, PCP degradation activity was more stable when a mixture of glucose and sodium formate was used as secondary carbon source instead of glucose. The analysis of the chlorophenol intermediates suggested that the main pathway of PCP dechlorination was PCP 2,3,5,6-tetrachlorophenol 2,3,5-trichlorophenol 3,5-dichlorophenol 3-chlorophenol phenol. In a laboratory-scale continuous-upflow fixed-film column reactor, a PCP removal of more than 99% was achieved at a PCP loading rate of 60 mol (1 reactor volume)^–1 day^–1 for a hydraulic retention time of 0.7 day. Analysis of culture samples taken at different levels in the reactor have shown that, at this PCP loading rate, only the lower part of the reactor was active. 3-chlorophenol and 3,5- and 3,4-dichlorophenol were detected at the different levels of the reactor. A study of the microorganisms in the biofilm was carried out by scanning electron microscopy and suggested that the microorganisms involved in the consortium were present as a well-structured arrangement. Methanosaeta-like microorganisms were observed mainly at the base of the biofilm whereas, at the surface, a larger diversity of morphotypes was observed in which coccoid or small rod organisms were dominant. This work shows the importance of the design and the control of the operation parameters on the efficiency of the fixed-film reactor.     

Isolation and characterization of a new bacterium carboxylating phenol to benzoic acid under anaerobic conditions.

A consortium of spore-forming bacteria transforming phenol to benzoic acid under anaerobic conditions was treated with antibiotics to eliminate the four Clostridium strains which were shown to be unable to accomplish this reaction in pure culture and coculture. Clostridium ghonii was inhibited by chloramphenicol (10 micrograms/ml), whereas Clostridium hastiforme (strain 3) and Clostridium glycolicum were inhibited by clindamycin (20 micrograms/ml), without the transformation of phenol being affected. Electron microscopic observations of resulting liquid subcultures revealed the presence of two different bacilli: a dominant C hastiforme strain (strain 2) (width, 1 micron) and an unidentified strain 6 (width, 0.6 micron) which was not detected on solid medium. Bacitracin (0.5 U/ml) changed the ratio of the strains in favor of strain 6. C hastiforme 2 was eliminated from this culture by dilution. The isolated strain 6 transformed phenol to benzoic acid and 4-hydroxybenzoic acid to phenol and benzoic acid in the presence of proteose peptone. Both of these activities are inducible. This strain is a gram- variable, flagellated rod with a doubling time of 10 to 11 h in the presence of phenol. It has a cellular fatty acid composition like that of C. hastiforme. However, strain 6 does not hydrolyze gelatin or produce indole. The 16S rRNA sequence of strain 6 was found to be most similar to that of some Clostridium species, with homology ranging from 80 to 86%. Tbe evolutionary relationships of strain 6 to different groups of Clostridium and Clostridium-related species revealed that it does not emerge from any of these groups. Strain 6 most likely belongs to a new species closely related to Clostridium species.     

Development of a complementing cell line and a system for construction of adenovirus vectors with E1 and E2a deleted.

Although adenovirus vectors offer many advantages, it would be desirable to develop vectors with improved expression and decreased toxicity. Toward this objective, an adenovirus vector system with deletion of both the El and E2a regions was developed. A 5.9-kb fragment of the adenovirus type 5 (Ad5) genome containing the E2a gene and its early and late promoters was transfected into 293 cells. A complementing cell line, designated 293-C2, expressed the E2a mRNA and protein and was found to complement the defect in Ad5 viruses with temperature-sensitive or deletion mutations in E2a. A deletion of 1.3 kb removing codons 40 to 471 of the 529 amino acids of E2a was introduced into plasmids for preparation of viruses and vectors. An Ad5 virus with disruption of the El gene and deletion of E2a grew on 293-C2 cells but not on 293 cells. Vectors with E1 and E2a deleted expressing Escherichia coli beta-galactosidase or human alpha1-antitrypsin were prepared and expressed the reporter genes after intravenous injection into mice. This vector system retains sequences in common between the complementing cell line and the vectors, including 3.4 kb upstream and 1.1 kb downstream of the deletion. These vectors have potential advantages of increased capacity for insertion of transgene sequences, elimination of expression of E2a, and possibly reduction in expression of other viral proteins. Although the titers of the vectors with deleted are about 10- to 30-fold below those of vectors with E2a wild-type regions, the former vectors are suitable for detailed studies with animals to evaluate the effects on host immune responses, on duration of expression, and on safety.     

Biochemical characterization of three mycobacterial ribosomal fractions

The induction of antituberculous immunity by crude ribosomal fractions isolated from Mycobacterium tuberculosis strain H37Ra, M. bovis strain BCG, and M. smegmatis was studied in CF-1 mice. Levels of antituberculous immunity similar to that induced by live BCG were induced by the BCG and H37Ra ribosomal fractions whereas that isolated from M. smegmatis was found to be inactive. Electrophoresis of the three ribosomal fractions in sodium dodecyl sulfate - polyacylamide gels followed by differential staining showed the two active ribosomal fractions to be similar in their proteins, carbohydrate-containing substances, and lipid profiles. The inactive smegmatis ribosomal fraction differed mainly from the active ones on the basis of its carbohydrate-containing substances profile and by the absence of lipids. The polysaccharides and the ribosomes present in the H37Ra ribosomal fractions were purified by affinity chromatography on concanavalin A - Sepharose 4B. Each purified preparation showed no or only low antituberculous activity when injected separately, but when mixed together a high protection was observed. The formation of complexes between the ribosomes and the polysaccharide fraction was suggested and appears to be necessary for the induction of antituberculous immunity.     

Cloning of cDNA for argininosuccinate synthetase mRNA and study of enzyme overproduction in a human cell line

Previous studies of the human cell line RPMI-2650 (wild type) and its canavanine-resistant variants have demonstrated differences in argininosuccinate synthetase activity as follows: canavanine-resistant much greater than wild type grown in citrulline greater than wild type grown in arginine (Su, T.-S., Beaudet, A. L., and O'Brien, W. E. (1981) Biochemistry 20, 2956-2960). A recombinant plasmid containing a 1.55-kilobase insert complementary to the mRNA for human argininosuccinate synthetase was isolated by the combined use of differential colony hybridization and immunoprecipitation of the products of plasmid-selected mRNA translation. Both blot and dot hybridization analysis of polyadenylated RNA indicated a major mRNA species of 1.67 kilobase in all cells, and the levels of mRNA correlated well with the levels of enzyme activity: canavanine-resistant, 180; wild type grown in citrulline, 7; and wild type grown in arginine, 1. One major mRNA species of 1.67 kilobase and one minor species of 2.68 kilobase were observed in wild type and canavanine-resistant cell lines. Reassociation kinetics of pAS1 with genomic DNA from human liver, canavanine-resistant cells, and wild type cells were not significantly different. Blot hybridization of genomic DNA revealed no detectable differences between wild type cells, canavanine-resistant cells, and human leukocytes. The data demonstrated that there were multiple copies, perhaps 10 or more, of argininosuccinate synthetase-like sequences in human DNA and that the canavanine-resistant phenotype was not due to gene amplification.     

Interference of Neisseria gonorrhoeae growth by aerobic bacterial representatives of the urogenital flora

Aerobic bacterial isolates obtained from endocervical, vaginal and urethral swabbings were tested for interference of neisseria gonorrhoeae growth on solid medium. Simultaneous antagonism was studied using the lawn spotting method, and delayed antagonism by the basal spot/lawn method. From 58 swabbings we recuperated a total of 181 isolates, 71 of those were found interfering with at least one out of four gonococcal strains (G-1, G-2, G-3 and G-4). Similar percentages of interfering isolates were obtained from each of the isolation sites. The identification of the interfering isolates has revealed that similar numbers of coagulase negative staphylococci and identical numbers of group D streptococci were found for each of those sites. The majority of the interfering isolates and also of the inhibitory coagulase negative staphylococci showed only simultaneous antagonism. To complete the interference spectrum, we have tested all the active urogenital isolates against four other gonococcal strains (G-7, G-9, G-10 and G-11). This spectrum showed clearly that interference is not an all or none phenomenon. While the gonococcal interference spectrum of most of the Gram positive cocci and the Acinetobacter sp. strains is broad, that of all the other isolates is relatively narrow. Gonococcal strains G-7 and G-9 were the most susceptible to inhibition by the interfering urogenital isolates while strain G-3 was the most resistant one.     

Benign missense variations in the cystic fibrosis gene.

The common mutation causing cystic fibrosis is a deletion of phenylalanine 508 (delta F508), which occurs in a putative nucleotide-binding fold of the gene product. We report two additional mutations, substitution of cysteine for phenylalanine 508 (F508C) and substitution of valine for isoleucine 506 (I506V). Three compound heterozygous persons, two delta F508/F508C and one delta F508/I506V, had normal clinical and epithelial physiological studies indicating that the F508C and I506V mutations are benign. This opportunity to study the in vivo function of these mutations suggests that amino acid substitutions are more benign than changes in the length of this portion of the putative nucleotide-binding fold. These mutations must be taken into account when performing molecular diagnosis and carrier detection for cystic fibrosis.     

Neurofilament gene expression in transgenic mice

1. DNA fragments that include the human neurofilament NF-L gene was found to be correctly expressed in the majority of neurons in transgenic mice. 2. The NF-L transgene product, which is detectable in situ with a species-specific monoclonal antibody, provides a powerful genotype marking system applicable to developmental and regeneration studies of the mammalian nervous system. 3. The proximal 5'-flanking region of the NF-L gene is sufficient to direct expression of a heterologous gene in the mouse nervous system.     

Linkage mapping and fluorescence in situ hybridization of TCTE1 on human chromosome 6p: Analysis of dinucleotide polymorphisms on native gels

Highly informative dinucleotide repeat polymorphisms were identified at the T-complex-associated-testes-expressed-1 (TCTE1) locus on human chromosome 6p. Electrophoresis of single-stranded DNA on native gels facilitated the analysis of the dinucleotide polymorphisms. Linkage mapping positions this marker midway between the centromere and HLA with recombination fractions as follows: D6Z1-0.21-TCTE1-0.24-HLA. Two-color fluorescence in situ hybridization places TCTE1 proximal to CRIL171 (D6S19). Together, linkage and in situ hybridization indicate that the order of the loci is D6Z1-D6S4-D6S90-TCTE1-D6S19-D6S29-HLA-telomere. A sequence tagged site (STS) was established, and three yeast artificial chromosome (YAC) clones were identified for the TCTE1 locus.