Screening for dioxin-degrading basidiomycetes from temperate and tropical forests

A total of 500 strains of basidiomycetes isolated from temperate forests in Japan and 379 strains from tropical forests in Indonesia were subjected to a laboratory screening for dioxin-degrading ability. At first, about 200 fungal strains were selected by their ability to decolorize Remazol Brilliant Blue R dye as an indicator of ligninolytic activities. Next, for excluding the factor of dioxin sorption by mycelia, we prepared two series of living cultures exposed either long-term or short-term to 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), and compared the decreases in the remaining amounts of this model compound. We chose Bjerkandera adusta strain VH57 as a promising new candidate for dioxin degradation, because it gave 40% difference in 2,7-DCDD levels between the two treatments after 30 days of exposure.     

Enhancement of biodegradation of 2,7-dichlorodibenzo-p-dioxin by addition of fungal culture filtrate

The hydrophilicity of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), a model dioxin compound, increased when incubated with the culture filtrates of several strains of fungi. The possibility that the addition of these filtrates could enhance the biodegradation of 2,7-DCDD by the white-rot basidiomycetous fungus Phanerochaete sordida YK-624 was examined. The decrease of 2,7-DCDD after 3 weeks incubation in a YK-624 culture containing these filtrates was greater (30%) than that in the culture of YK-624 alone (15%). This is the first report describing the enhancement of dioxin decrease by the addition of a fungal filtrate.     

Biodegradation of diphenyl ethers by a copper-resistant mutant ofErwinia sp.

Summary A bacterium tentatively identified as anErwinia sp. was isolated from sewage by enrichment on methanol and lignin. Several mutants developed from this strain were studied for their ability to degrade aromatic ethers. Different concentrations of the chemicals were incubated with the organisms and the degradation was estimated by high-performance liquid chromatography (HPLC). Among these mutants, one isolate,Erwinia sp. strain CU3614, showed resistance to copper ions (>20 mM CuSO₄) and the ability to degrade 4-hydroxydiphenyl ether (4-HDPE), 4-chlorodiphenyl ether (4-CDPE), 4-nitrodiphenyl ether (4-NDPE) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) in the presence of copper ions. Increased concentrations of copper in the medium resulted in higher degradation of 4-HDPE. Further studies with copper-sensitive mutants obtained fromErwinia sp. CU3614 by Tn5 transposon-induced mutagenesis showed a corresponding decrease in the ability to degrade 4-HDPE. These results suggest the presence of a copper-associated activity in the biotransformation of aromatic ethers.     

Biotransformation of polychlorinated dibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin by <Emphasis Type="Italic">Coprinellus</Emphasis> species

A degradation experiment on dibenzo-p-dioxin (DD) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) was carried out using basidiomycetous fungi belonging to the genera Coprinus, Coprinellus, and Coprinopsis. Some species showed a high rate of decrease in DD for the 2-week test period. Among them, Coprinellus disseminatus showed the highest ability to decrease the DD level, close to 100% by the end of 2 weeks. Further examination showed that Coprinellus disseminatus and Coprinellus micaceus, belonging to the genus Coprinellus, were able to metabolize 2,7-DCDD to a monohydroxylated compound, probably mediated by the P450 system. The metabolism of chlorinated DD by fungi capable of living in soil conditions is reported here for the first time.     

Intron-dependent accumulation of mRNA in Coriolus hirsutus of lignin peroxidase gene the product of which is involved in conversion/degradation of polychlorinated aromatic hydrocarbons

The homobasidiomycete Coriolus hirsutus coding sequences of a lignin peroxidase (LiP) gene (lip, containing six (I-VI) introns), a lip cDNA (lipc), and three lipc derivatives containing one (I), three (I-III), or five (I-V) introns were inserted into chromosome-integrating expression vector. These recombinant plasmids were introduced into C. hirustus monokaryotic strain. The transformant carrying the promoter-lipc-terminator cassette did not contain enough mRNA molecules to be detectable by Northern-blot analysis. On the other hand, all the transformants carrying cassettes of genomic lip and intron(s)-containing lipc sequences contained sufficient amounts of mRNAs to be easily detected by Northern-blot analysis. LiP activities in the culture supernatants of these transformants were found to be about five times as high as those of transformants carrying the lipc cassette (or no cassette). The culture supernatants of the transformants with high LiP activity showed remarkably high conversion activity toward pentachlorophenol (PCP) and degradation activity toward 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). These results indicate that at least one intron (intron I) is required for accumulation of lip mRNA and its subsequent translational expression in C. hirsutus.     

Relative ability of two strains of Pucccinia graminis tritici to survive when mixed

The relationship between relative survival ability and the number of genes for virulence in two strains of Puccinia graminis tritici was investigated using a differential variety to determine the proportion of each race in the mixture. The strain with the widest host range (21 Anz-2,3,7) became predominant after a number of uredial generations, regardless of its proportion in the original inoculum mixture. However, despite the higher relative survival ability of strain 21 Anz-2,3,7, strain 21 Anz-2,7 persisted at c . 1% frequency after four successive uredial generations. The longer the period between successive inoculations, the more rapidly strain 21 Anz-2,3,7 became predominant in the mixture. The variety on which the mixture was cultured did not influence the relative survival ability of the two strains.     

The growth advantage in stationary-phase (GASP) phenomenon in mixed cultures of enterobacteria

Growth advantage in stationary phase (GASP) is the term used to describe the ability of mutants with an increased fitness from 10-day-old enterobacteria culture to out-compete 1-day-old cells of the same initial strain during a prolonged stationary phase, although the aged cells are introduced as a minority. We studied this bacterial trait in mixed cultures of two enterobacterial species, Escherichia coli and Salmonella enterica, wild type in addition to derived mutants from both strains that contain chromosomal-encoded resistance to either nalidixic acid or streptomycin. The strong GASP phenotype was obtained in mixed cultures with the aged mutant strains, but not when the isogenic antibiotic-sensitive strains were used. This phenomenon was associated with chromosomal rearrangements in 10-day-old bacterial antibiotic-resistant mutated cells.     

Phylogenetical approach to isolation of white-rot fungi capable of degrading polychlorinated dibenzo-p-dioxin

A degradation experiment on PCDDs and phylogenetical analyses were carried out on newly isolated 2,7-dichlorodibenzo-p-dioxin (2,7-diCDD)-degrading white-rot fungi, strains BMC3014, BMC9152, and BMC9160. When these fungi were incubated with tri- or tetraCDDs, the substrates were degraded efficiently, and hydroxylated metabolites were detected. On the other hand, 1,3,6,8-tetrachlorodibenzo-p-dioxin was not decreased, and no metabolites were detected. Phylogenetic analysis of internal transcribed spacers (ITSs) containing rRNA gene sequence (ITS-rDNA) clarified that these strains belonged to the genus Phlebia and were closely related to the fungi Phlebia lindtneri, strains MZ-227 and MG-60, which had both been isolated as 2,7-diCDD-degrading fungi in our previous study. Based on this phylogenetical relationship, other Phlebia genera species were used for a degradation experiment on 2,7-diCDD and 1,3,6,8-tetraCDD. Phlebia acerina and Phlebia brevispora degraded 2,7-diCDD about 40 and 80%, respectively, over 14 days of incubation. It became clear that P. brevispora can degrade 1,3,6,8-tetraCDD and transform it to monohydroxy-tetraCDD, monomethoxy-tetraCDD, dimethoxy-tetraCDD, dimethoxy-triCDD, and 3,5-dichlorocatechol in the treatment cultures. In this paper, we could clearly prove for the first time by identifying the metabolites that white-rot fungus P. brevispora could degrade the recalcitrant dioxin, 1,3,6,8-tetraCDD.     

Isolation and characterization of dibenzofuran-degrading bacteria

Two bacterial strains capable of utilizing dibenzofuran (DF) as a sole carbon source were isolated from soil samples of reclaimed land. The strains designated HL1 and HL7 were identified as Klebsiella sp. and Sphingomonas sp., respectively, on the basis of biochemical characteristics and the sequences of the 16S ribosomal DNA. Sphingomonas sp. strain HL7 degraded non-, mono- and also dichlorinated DF and dibenzo-p-dioxin (DD). Klebsiella sp. strain HL1 was able to degrade non- and monochlorinated DFs and DDs, but not dichlorinated ones. The metabolites formed from DF by strains HL1 and HL7 were similar to those by dioxin-degrading bacteria Sphingomonas sp. strain RW1 except for salicylic acid and catechol. Strain HL7 had a gene homologous to that encoding the dioxin dioxygenase alpha-subunit (dxnA1) gene of Sphingomonas sp. strain RW1. However, Southern hybridization analysis showed that the size of an EcoRV-digested genomic fragment involving the dioxin dioxygenase gene of strain HL7 was smaller than that of strain RW1, and that strain HL1 did not have the homologous gene. Strains HL1 and HL7 provided useful information regarding the dioxygenase genes.     

Macrophages exposed to lymphotropic and monocytotropic HIV induce similar CTL responses despite differences in productive infection

Macrophages are accessory cells that are vulnerable to infection by HIV-1. HTLV-IIIB, a lymphotropic strain of HIV, infects macrophages poorly resulting in either no or low levels of virus expression compared to high levels of productive infection after exposure of macrophages to the monocytotropic HIV strain Ada-M. Whether this results in an impaired ability of HTLV-IIIB-exposed macrophages to initiate protective cytotoxic T lymphocyte (CTL) immune responses against these strains is not well defined. We investigated the ability of monocyte-derived macrophages (MDM) exposed to lymphotropic and monocytotropic HIV strains to initiate primary CTL responses in vitro. MDM exposed to HTLV-IIIB induced a specific primary CTL response that was comparable to MDM exposed to the monocytotropic strain Ada-M despite marked differences in productive HIV infection in MDM between the two strains. CTL generated in this model were MHC-restricted, strain-specific, and CD8+. These data demonstrate that high levels of productive HIV infection in accessory cells are not a prerequisite for the generation of a primary CTL response, suggesting a novel immunologic interaction between MDM and lymphotropic HIV strains.     

Catabolic characteristics of biphenyl-utilizing isolates which cometabolize PCBs

As there are at least three types of bacteria involved in the aerobic mineralization of polychlorinated biphenyls (PCBs), this study was undertaken to determine what catabolic features are lacking in biphenyl-degraders and to determine if chlorobenzoate- and chloroacetate-utilizing bacteria are as indigenous to soil as biphenyl-degraders. Bacteria were tested for their ability to utilize chlorinated acids and to cometabolize Aroclor 1254 and dibenzo-p-dioxane (dioxin). The broad and variable substrate specificity of the biphenyl dioxygenase among strains was noted by the range of <1 to 53% cometabolism of total PCB congeners and by the oxidation of dioxin, which was not a growth substrate. Growth on chloroalkanoic acids was more frequent with 2-chloropropionate (87% of all strains), 3-chloropropionate (72%), 4-chlorobutyrate (66%), and less frequent (28%) withtrans-3-chlorocrotonate. However, only one strain,Pseudomonas fluorescens K3, could utilize chloroacetate. No biphenyl-utilizers grew on 2- or 4-chlorobenzoate, and only five strains grew on 3-chlorobenzoate. Acetate and benzoate-utilizers were found in all three soils tested at levels near 10⁶/g, whereas chloroacetate- or chlorobenzoate-utilizers were not detected. The inability of biphenyl-degraders to dehalogenate the products of PCB cometabolism is clearly unrelated to metabolism of saturated chloroaliphatic acids, with the notable exception of chloroacetate, since most strains grew on them. Thus, the inability to utilize chloroacetate, a central intermediate in the meta fission pathway, may be relevant to the incomplete catabolism of PCBs by biphenyl-utilizers.     

Enhanced Resistance to Nitrosoguanidine Killing and Mutagenesis in a DNA Gyrase Mutant of Escherichia coli

The role of DNA gyrase in handling DNA damages induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined with two Escherichia coli strains, KL161 and KL166. The two strains are isogenic except that KL166 harbors a mutation at the nalA (gyrA) locus which specifies one of the two subunits of DNA gyrase. We treated the two strains with several different types of mutagenic agents and found the nalA strain to be highly resistant to MNNG-induced killing and mutagenic effects as compared with the parental strain. The MNNG resistance was specific, since the two strains were about equally sensitive to methyl methane sulfonate, ethyl methane sulfonate, and UV and gamma radiations. We pulse-labeled the two strains with [(3)H]uridine and (14)C-amino acids after MNNG treatment to analyze RNA and protein synthetic rates. The pulse-labeled proteins were also separated on polyacrylamide gels. The results show that pulse-labeled RNA and proteins persisted in the nalA strain but declined rapidly in the parental strain after MNNG treatment. We compared membrane-free nucleoid preparations from the two strains by sucrose density gradient centrifugation and found a difference in nucleoid organization between the two strains. The nucleoid of the nalA strain, unlike that of the parental strain, may have a highly ordered structure, as indicated by its resistance to ethidium bromide-induced relaxation. The ability of the two strains to express an adaptive response to MNNG was determined. We found that the resistance to MNNG killing and mutagenesis by the nalA strain cannot be further increased by adaptive treatment. These results suggest that an alteration in DNA gyrase may have profound effects on E. coli chromosome organization and base methylation by MNNG.     

Properties of inhibitors of plant virus infection occurring in the leaves of species in the Chenopodiales

The process of infection by strains 21 Anz-2, 7 and 21 Anz-2, 3, 7 of Puccinia graminis tritici was quantitatively investigated to determine whether differences existed between them at any stage of the infection process and whether any such differences were related to the relative survival ability of the two strains in mixtures. The frequency of urediospore germination, the frequency of formation of appressoria, the period between inoculation and the eruption of uredia, the proportion of infection courts developing into uredia, and the visible characteristics of the uredia, did not differ between the two strains. However, penetration occurred more frequently from appressoria of strain 21 Anz-2, 3, 7 than from those of strain 21 Anz-2, 7. Uredia of strain 21 Anz-2,3, 7 increased more rapidly in size than did those of 21 Anz-2,7 and were ultimately larger in area. Similarly, more urediospores were produced per uredium by strain 21 Anz-2, 3, 7 than by 21 Anz-2, 7. It is suggested that these differences in frequency of penetration and in number of urediospores per uredium could account, at least in part, for the difference in relative survival ability between the two strains.     

Description of a bacteriocinogenic plasmid in Beneckea harveyi.

A total of 795 strains of marine Vibrio species and Beneckea harveyi, a luminescent marine bacterium, were isolated from various sources in the area of Galveston Island, Tex., and screened for the production of bacteriocin-like substances. More than 8% of the Vibrio isolates produced low-molecular-weight (dialyzable) substances, which were lethal to a test strain of V. parahaemolyticus. Approximately 5% of the B. harveyi isolates produced higher-molecular-weight (nondialyzable) substances which were lethal to a test strain of B. harveyi. One of the B. harveyi strains (strain SY) produced a nondialyzable substance which was lethal to two of 39 strains of B. harveyi. The substance showed no activity toward 17 test strains drawn from the Vibrionaceae and Enterobacteriaceae. Strain SY showed no sensitivity to its own lethal agent and was shown by agarose gel electrophoresis and electron microscopy to harbor a single plasmid of 38 x 10(6) daltons. Variants of strain SY lacking the plasmid were produced by growth in the presence of the antibiotic novobiocin. These variants lacked both the ability to produce the lethal substance and the ability to survive in its presence. The lethal agent produced by strain SY is the first bacteriocin reported in marine bacteria. The term "harveyicin" is proposed to name this lethal substance.     

Microbial oxidation of dimethylnaphthalene isomers.

Three bacterial strains, identified as Alcaligenes sp. strain D-59 and Pseudomonas sp. strains D-87 and D-186, capable of growing on 2,6-dimethylnaphthalene (2,6-DMN) as the sole source of carbon and energy were isolated from soil samples. 2,6-Naphthalene dicarboxylic acid was formed in the culture broths of these three strains grown on 2,6-DMN. In addition, 2-hydroxymethyl-6-methylnaphthalene and 6-methylnaphthalene-2-carboxylic acid were detected in the culture broth of strain D-87. Strain D-87 grew well on 1,2-, 1,3-, 1,4-, 1,5-, 2,3-, and 2,7-DMN as the sole source of carbon and energy and accumulated 2-methylnaphthalene-3-carboxylic acid and 2,3-naphthalene dicarboxylic acid from 2,3-DMN, 4-methylnaphthalene-1-carboxylic acid from 1,4-DMN, and 7-methylnaphthalene-2-carboxylic acid from 2,7-DMN.     

Pathogenic Potential of Saccharomyces Strains Isolated from Dietary Supplements

Saccharomyces cerevisiae plays a beneficial role in health because of its intrinsic nutritional value and bio-functional properties, which is why it is also used as a dietary supplement. However, the perception that S. cerevisiae is harmless has changed due to an increasing number of infections caused by this yeast. Given this scenario, we have tested whether viable strains contained in dietary supplements displayed virulence-associated phenotypic traits that could contribute to virulence in humans. We have also performed an in vivo study of the pathogenic potential of these strains using a murine model of systemic infection by intravenous inoculation. A total of 5 strains were isolated from 22 commercial products and tested. Results highlight one strain (D14) in terms of burden levels in brains and kidneys and ability to cause death, whereas the other two strains (D2 and D4) were considered of low virulence. Our results suggest a strong relationship between some of the virulence-associated phenotypic traits (ability to grow at 39°C and pseudohyphal growth) and the in vivo virulence in a mouse model of intravenous inoculation for isolates under study. The isolate displaying greatest virulence (D14) was evaluated in an experimental murine model of gastrointestinal infection with immunosuppression and disruption of mucosal integrity, which are common risk factors for developing infection in humans, and results were compared with an avirulent strain (D23). We showed that D14 was able to spread to mesenteric nodes and distant organs under these conditions. Given the widespread consumption of dietary supplements, we recommend only safe strains be used.     

Physiological bases of genetic differences in cannibalism behavior of the confused flour beetle Tribolium confusum

Physiological causes of genetic differences in cannibalism were examined to gain a better understanding of constraints on behavior evolution. Cannibalism has complex population level consequences in Tribolium confusum, including dramatic effects on population size. Laboratory strains with low and high cannibalism rates, obtained through inbreeding, have maintained distinct levels of cannibalism for over two decades even in the absence of artificial selection to maintain the differences. Why strains differ in their cannibalism rates was examined by measuring: (1) the nutritional benefit from cannibalism in both nutritionally good and poor environments, and (2) the possibility that eggs are an important source of water. How strains achieve differences in cannibalism was examined by testing for differences between strains in their ability to find eggs and in their tendency to eat eggs. Beetles from both strains survive equally well in a nutritionally good environment, but they accomplish this in different ways. The low cannibalism strain has high survivorship with and without cannibalism. The high cannibalism strain has low survivorship when not fed eggs and survivorship equivalent to the low cannibalism strain when fed eggs, suggesting it compensates for poor nutritional adaptation by eating eggs. The strains also differ in feeding behavior; beetles from the high cannibalism strain have a higher appetite for eggs. Beetles from the two strains did not differ in locomotor activity, search efficiency, or need for water. The observed behavioral and nutritional differences may contribute to the maintenance of different levels of cannibalism.