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R T Lamar

Publications and source records attributed to R T Lamar.

12 recordsLinked to original sources
PubMed1996

Expression of lip genes during growth in soil and oxidation of anthracene by Phanerochaete chrysosporium.

mRNA extraction from soil and quantitation by competitive reverse transcription-PCR were combined to study the expression of the 10 known lignin peroxidase (lip) genes in anthracene-transforming soil cultures of Phanerochaete chrysosporium. Levels of extractable lipA transcript and protein (LiP H8) were well correlated, although they were separated by a 2-day lag period. The patterns of transcript abundance over time in soil-grown P. chrysosporium varied among the nine lip mRNAs detected; comparison with lip gene expression under different liquid culture conditions suggested an early phase of carbon limitation for the cultures as a whole, which was followed by a transition to nitrogen starvation. Anthracene transformation occurred throughout the 25-day course of the experiment and, therefore, likely involves mechanisms distinct from those involved in oxidation of non-LiP substrate polycyclic aromatic hydrocarbons.

Anthracenes↗
PubMed1996

Polycyclic aromatic hydrocarbon-degrading capabilities of Phanerochaete laevis HHB-1625 and its extracellular ligninolytic enzymes.

The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs) in liquid culture was studied in relation to its complement of extracellular ligninolytic enzymes. In nitrogen-limited liquid medium, P. laevis produced high levels of manganese peroxidase (MnP). MnP activity was strongly regulated by the amount of Mn2+ in the culture medium, as has been previously shown for several other white rot species. Low levels of laccase were also detected. No lignin peroxidase (LiP) was found in the culture medium, either by spectrophotometric assay or by Western blotting (immunoblotting). Despite the apparent reliance of the strain primarily on MnP, liquid cultures of P. laevis were capable of extensive transformation of anthracene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Crude extracellular peroxidases from P. laevis transformed all of the above PAHs, either in MnP-Mn2+ reactions or in MnP-based lipid peroxidation systems. In contrast to previously published studies with Phanerochaete chrysosporium, metabolism of each of the four PAHs yielded predominantly polar products, with no significant accumulation of quinones. Further studies with benz[a]anthracene and its 7,12-dione indicated that only small amounts of quinone products were ever present in P. laevis cultures and that quinone intermediates of PAH metabolism were degraded faster and more extensively by P. laevis than by P. chrysosporium.

Basidiomycota↗
PubMed1996

Fluorene Oxidation In Vivo by Phanerochaete chrysosporium and In Vitro during Manganese Peroxidase-Dependent Lipid Peroxidation.

The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin peroxidase, was studied in liquid cultures of Phanerochaete chrysosporium and in vitro with P. chrysosporium extracellular enzymes. Intact fungal cultures metabolized fluorene to 9-hydroxyfluorene via 9-fluorenone. Some conversion to more-polar products was also observed. Oxidation of fluorene to 9-fluorenone was also obtained in vitro in a system that contained manganese(II), unsaturated fatty acid, and either crude P. chrysosporium peroxidases or purified recombinant manganese peroxidase. The oxidation of fluorene in vitro was inhibited by the free-radical scavenger butylated hydroxytoluene but not by the lignin peroxidase inhibitor NaVO(inf3). Manganese(III)-malonic acid complexes could not oxidize fluorene. These results indicate that fluorene oxidation in vitro was a consequence of lipid peroxidation mediated by P. chrysosporium manganese peroxidase. The rates of fluorene and diphenylmethane disappearance in vitro were significantly faster than those of true polycyclic aromatic hydrocarbons or fluoranthenes, whose rates of disappearance were ionization potential dependent. This result indicates that the initial oxidation of fluorene proceeds by mechanisms other than electron abstraction and that benzylic hydrogen abstraction is probably the route for oxidation.

Journal Article↗
PubMed1996

Development of fungal inocula for bioaugmentation of contaminated soils.

This report describes novel fungal inocula for bioaugmentation of soils contaminated with hazardous organic compounds. The inocula are in the form of pelleted solid substrates coated with a sodium alginate suspension of fungal spores or mycelial fragments and incubated until overgrown with the mycelium of selected lignin-degrading fungi. The organisms evaluated were Phanerochaete chrysosporium (BKM F-1767, ATCC 42725), P. sordida (HHB-8922-Sp), Irpex lacteus (Mad-517, ATCC 11245), Bjerkandera adusta (FP-135160-Sp, ATCC 62023), and Trametes versicolor (MD-277). The pelleted fungal inocula resisted competition and proliferation from indigenous soil microbes, were lower in moisture content than current fungal inocula, and had sufficient mechanical strength to allow handling and introduction into the soil without a change in the mechanical consistency of the pellets. Inoculated at a rate of 3% in artificially contaminated nonsterile soil, I. lacteus, B. adusta, and T. versicolor removed 86, 82, and 90%, respectively, of the pentachlorophenol in 4 weeks. A mathematical model was developed to explain moisture distribution in a hydrogel-coated pelleted substrate.

Journal Article↗
PubMed1996

Manganese peroxidase mRNA and enzyme activity levels during bioremediation of polycyclic aromatic hydrocarbon-contaminated soil with Phanerochaete chrysosporium.

mRNA extraction from soil and quantitation by competitive reverse transcription-PCR were combined to study the expression of three manganese peroxidase (MnP) genes during removal of polycyclic aromatic hydrocarbons from cultures of Phanerochaete chrysosporium grown in presterilized soil. Periods of high mnp transcript levels and extractable MnP enzyme activity were temporally correlated, although separated by a short (1- to 2-day) lag period. This time frame also coincided with maximal rates of fluorene oxidation and chrysene disappearance in soil cultures, supporting the hypothesis that high ionization potential polycyclic aromatic hydrocarbons are oxidized in soil via MnP-dependent mechanisms. The patterns of transcript abundance over time in soil-grown P. chrysosporium were similar for all three of the mnp mRNAs studied, indicating that transcription of this gene family may be coordinately regulated under these growth conditions.

Base Sequence↗
PubMed1995

Quantitation of fungal mRNAs in complex substrates by reverse transcription PCR and its application to Phanerochaete chrysosporium-colonized soil.

Thorough analysis of fungi in complex substrates has been hampered by inadequate experimental tools for assessing physiological activity and estimating biomass. We report a method for the quantitative assessment of specific fungal mRNAs in soil. The method was applied to complex gene families of Phanerochaete chrysosporium, a white-rot fungus widely used in studies of organopollutant degradation. Among the genes implicated in pollutant degradation, two closely related lignin peroxidase transcripts were detected in soil. The pattern of lignin peroxidase gene expression was unexpected; certain transcripts abundant in defined cultures were not detected in soil cultures. Transcripts encoding cellobiohydrolases and beta-tubulin were also detected. The method will aid in defining the roles of specific genes in complex biological processes such as organopollutant degradation, developing strategies for strain improvement, and identifying specific fungi in environmental samples.

Base Sequence↗
PubMed1995

One-electron oxidation in the degradation of creosote polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium.

The abilities of whole cultures of Phanerochaete chrysosporium and P. chrysosporium manganese peroxidase-mediated lipid peroxidation reactions to degrade the polycyclic aromatic hydrocarbons (PAHs) found in creosote were studied. The disappearance of 12 three- to six-ring PAHs occurred in both systems. Both in vivo and in vitro, the disappearance of all PAHs was found to be very strongly correlated with ionization potential. This was true even for compounds beyond the ionization potential thresholds of lignin peroxidase and Mn3+. Deviations from this correlation were seen in the cases of PAHs which are susceptible to radical addition reactions. These results thus begin to clarify the mechanisms of non-lignin peroxidase-labile PAH degradation in the manganese peroxidase-lipid peroxidation system and provide further evidence for the ability of this system to explain the in vivo oxidation of these compounds.

Basidiomycota↗
PubMed1994

Manganese peroxidases of the white rot fungus Phanerochaete sordida.

The ligninolytic enzymes produced by the white rot fungus Phanerochaete sordida in liquid culture were studied. Only manganese peroxidase (MnP) activity could be detected in the supernatant liquid of the cultures. Lignin peroxidase (LiP) and laccase activities were not detected under a variety of different culture conditions. The highest MnP activity levels were obtained in nitrogen-limited cultures grown under an oxygen atmosphere. The enzyme was induced by Mn(II). The initial pH of the culture medium did not significantly affect the MnP production. Three MnP isozymes were identified (MnPI, MnPII, and MnPIII) and purified to homogeneity by anion-exchange chromatography followed by hydrophobic chromatography. The isozymes are glycoproteins with approximately the same molecular mass (around 45 kDa) but have different pIs. The pIs are 5.3, 4.2, and 3.3 for MnPI, MnPII, and MnPIII, respectively. The three isozymes are active in the same range of pHs (pHs 3.0 to 6.0) and have optimal pHs between 4.5 and 5.0. Their amino-terminal sequences, although highly similar, were distinct, suggesting that each is the product of a separate gene.

Amino Acid Sequence↗
PubMed1990

Selective Medium for Isolating Phanerochaete chrysosporium from Soil.

A selective medium was developed that is capable of isolating Phanerochaete chrysosporium from soil. This medium contains 15 ppm of benomyl (15 mug g) and 550 ppm of streptomycin sulfate in 2% malt agar and is held at 39 degrees C after inoculation. P. chrysosporium was isolated from three nonsterile forest soils to which the fungus had been added. These soils contained large microbial populations.

Journal Article↗
PubMed1990

In Situ Depletion of Pentachlorophenol from Contaminated Soil by Phanerochaete spp.

The ability of two white rot fungi to deplete pentachlorophenol (PCP) from soil, which was contaminated with a commercial wood preservative, was examined in a field study. Inoculation of soil containing 250 to 400 mug of PCP g with either Phanerochaete chrysosporium or P. sordida resulted in an overall decrease of 88 to 91% of PCP in the soil in 6.5 weeks. This decrease was achieved under suboptimal temperatures for the growth and activity of these fungi, and without the addition of inorganic nutrients. Since the soil had a very low organic matter content, peat was included as a source of organic carbon for fungal growth and activity. A small percentage (8 to 13%) of the decrease in the amount of PCP was a result of fungal methylation to pentachloroanisole. Gas chromatographic analysis of sample extracts did not reveal the presence of extractable transformation products other than pentachloroanisole. Thus, when losses of PCP via mineralization and volatilization were negligible, as they were in laboratory-scale studies (R. T. Lamar, J. A. Glaser, and T. K. Kirk, Soil Biol. Biochem. 22:433-440, 1990), most of the PCP was converted to nonextractable soil-bound products. The nature, stability, and toxicity of soil-bound transformation products, under a variety of conditions, must be elucidated before use of these fungi in soil remediation efforts can be considered a viable treatment method.

Journal Article↗
PubMed1990

Sensitivity to and Degradation of Pentachlorophenol by Phanerochaete spp.

This research measured mycelial extension rates of selected strains of Phanerochaete chrysorhiza, Phanerochaete laevis, Phanerochaete sanguinea, Phanerochaete filamentosa, Phanerochaete sordida, Inonotus circinatus, and Phanerochaete chrysosporium and the ability of these organisms to tolerate and degrade the wood preservative pentachlorophenol (PCP) in an aqueous medium and in soil. Most of the tested species had mycelial extension rates in the range of </=0.5 to 1.5 cm day, but there were large interspecific differences. A notable exception, P. sordida, grew very rapidly, with an average mycelial extension rate of 2.68 cm day at 28 degrees C. Rank of species by growth rate was as follows: P. chrysosporium > P. sordida > P. laevis > P. chrysorhiza = P. sanguinea > I. circinatus = P. filamentosa. There were also significant intraspecific differences in mycelial extension rates. For example, mycelial extension rates among strains of P. sordida ranged from 1.78 to 4.81 cm day. Phanerochaete spp. were very sensitive to PCP. Growth of several species was prevented by the presence of 5 ppm (5 mug/g) PCP. However, P. chrysosporium and P. sordida grew at 25 ppm PCP, albeit at greatly decreased mycelial extension rates. In an aqueous medium, mineralization of PCP by P. sordida 13 (ca. 12% after 30 days) was significantly greater than that by all other tested P. sordida strains and P. chrysosporium. After 64 days, the level of PCP had decreased by 96 and 82% in soil inoculated with P. chrysosporium and P. sordida, respectively. Depletion of PCP by these fungi occurred in a two-stage process. The first stage was characterized by a rapid depletion of PCP that coincided with an accumulation of pentachloroanisole (PCA). At the end of the first stage, ca. 64 and 71% of the PCP was converted to PCA in P. chrysosporium and P. sordida cultures, respectively. In the second stage, levels of PCP and PCA were reduced by 9.6 and 18%, respectively, in soil inoculated with P. chrysosporium and by 3 and 23%, respectively, in soil inoculated with P. sordida. PCA was mineralized by both P. chrysosporium and P. sordida in an aqueous medium.

Journal Article↗