Methodological issues in biomonitoring of low level exposure to benzene.
Data from a pilot study on unmetabolized benzene and trans,trans muconic acid (t,t-MA) excretion in filling station attendants and unexposed controls were used to afford methodological issues in the biomonitoring of low benzene exposures (around 0.1 ppm). Urinary concentrations of benzene and t,t-MA were measured by dynamic head-space capillary GC/FID and HPLC, respectively. The accuracy of the HPLC determination of t,t-MA was assessed in terms of inter- and intra-method reliability. The adequacy of urinary t,t-MA and benzene as biological markers of low benzene exposure was evaluated by analysing the relationship between personal exposure to benzene and biomarker excretion. Filling station attendants excreted significantly higher amounts of benzene, but not of t,t-MA, than controls. Adjusting for occupational benzene exposure, smokers excreted significantly higher amounts of t,t-MA, but not of unmetabolized benzene, than nonsmokers. A comparative analysis of the present and previously published biomonitoring surveys showed a good inter-study agreement regarding the amount of t,t-MA and unmetabolized benzene excreted (about 0.1-0.2 mg/l and 1-2 micrograms/l, respectively) per unit of exposure (0.1 ppm). For each biomarker, based on the distribution of parameters observed in the pilot study, we calculated the minimum sample size required to estimate the population mean with given confidence and precision. (+info)
The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases and catalyzes aromatic monohydroxylation of benzene, toluene, and phenol.
The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase alpha-subunit (XamoA); aamB, encoding the 88-residue oxygenase gamma-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase beta-subunit (XamoE); and aamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of sigma54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase alpha-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase from Rhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacter strain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture of o-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studies Xanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of the aam gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol. (+info)
Evaluation of mycobacillin and versicolin as agricultural fungicides. II. Stability in soil.
The effect of paddy soils on mycobacillin and versicolin was investigated. Soil inactivated mycobacillin as determined by spectral analysis and microbiological assay. Soil can inactive mycobacillin only at or above the threshold concentration (125 approximately 130 mug per 10 mg of soil), the excess being unreacted. No new peak appears in the ultraviolet spectrum (240 approximately 300 nm) while mycobacillin is inactivated. Soil is without any effect on versicolin. (+info)
Adenosylcobalamin-mediated methyl transfer by toluate cis-dihydrodiol dehydrogenase of the TOL plasmid pWW0.
We identified and characterized a methyl transfer activity of the toluate cis-dihydrodiol (4-methyl-3,5-cyclohexadiene-cis-1, 2-diol-1-carboxylic acid) dehydrogenase of the TOL plasmid pWW0 towards toluene cis-dihydrodiol (3-methyl-4,5-cyclohexadiene-cis-1, 2-diol). When the purified enzyme from the recombinant Escherichia coli containing the xylL gene was incubated with toluene cis-dihydrodiol in the presence of NAD+, the end products differed depending on the presence of adenosylcobalamin (coenzyme B12). The enzyme yielded catechol in the presence of adenosylcobalamin, while it gave 3-methylcatechol in the absence of the cofactor. Adenosylcobalamin was transformed to methylcobalamin as a result of the enzyme reaction, which indicates that the methyl group of the substrate was transferred to adenosylcobalamin. Other derivatives of the cobalamin such as aquo (hydroxy)- and cyanocobalamin did not mediate the methyl transfer reaction. The dehydrogenation and methyl transfer reactions were assumed to occur concomitantly, and the methyl transfer reaction seemed to depend on the dehydrogenation. To our knowledge, the enzyme is the first dehydrogenase that shows a methyl transfer activity as well. (+info)
Molecular analysis of microbial community structures in pristine and contaminated aquifers: field and laboratory microcosm experiments.
This study used phylogenetic probes in hybridization analysis to (i) determine in situ microbial community structures in regions of a shallow sand aquifer that were oxygen depleted and fuel contaminated (FC) or aerobic and noncontaminated (NC) and (ii) examine alterations in microbial community structures resulting from exposure to toluene and/or electron acceptor supplementation (nitrate). The latter objective was addressed by using the NC and FC aquifer materials for anaerobic microcosm studies in which phylogenetic probe analysis was complemented by microbial activity assays. Domain probe analysis of the aquifer samples showed that the communities were predominantly Bacteria; Eucarya and Archaea were not detectable. At the phylum and subclass levels, the FC and NC aquifer material had similar relative abundance distributions of 43 to 65% beta- and gamma-Proteobacteria (B+G), 31 to 35% alpha-Proteobacteria (ALF), 15 to 18% sulfate-reducing bacteria, and 5 to 10% high G+C gram positive bacteria. Compared to that of the NC region, the community structure of the FC material differed mainly in an increased abundance of B+G relative to that of ALF. The microcosm communities were like those of the field samples in that they were predominantly Bacteria (83 to 101%) and lacked detectable Archaea but differed in that a small fraction (2 to 8%) of Eucarya was detected regardless of the treatment applied. The latter result was hypothesized to reflect enrichment of anaerobic protozoa. Addition of nitrate and/or toluene stimulated microbial activity in the microcosms, but only supplementation of toluene alone significantly altered community structures. For the NC material, the dominant subclass shifted from B+G to ALF, while in the FC microcosms 55 to 65% of the Bacteria community was no longer identifiable by the phylum or subclass probes used. The latter result suggested that toluene exposure fostered the proliferation of phylotype(s) that were otherwise minor constituents of the FC aquifer community. These studies demonstrated that alterations in aquifer microbial communities resulting from specific anthropogenic perturbances can be inferred from microcosm studies integrating chemical and phylogenetic probe analysis and in the case of hydrocarbon contamination may facilitate the identification of organisms important for in situ biodegradation processes. Further work integrating and coordinating microcosm and field experiments is needed to explore how differences in scale, substrate complexity, and other hydrogeological conditions may affect patterns observed in these systems. (+info)
Tremor induced by toluene misuse successfully treated by a Vim thalamotomy.
A 22 year old man developed a vigorous tremor of 5 Hz in his right hand, after a 7 year history of toluene misuse. T2 Weighted MRI depicted marked decreases in the signal intensity of the basal ganglia, red nucleus, and thalamus on both sides. The stereotactic coagulation of the left nucleus ventrointermedius (Vim) of the thalamus abolished the tremors in his right hand. This patient clearly exhibited the pathological involvement of rubral lesions in generation of a toluene induced tremor on MRI. Toluene induced tremor is an irreversible symptom which persists even after stopping toluene misuse, therefore in medically intractable cases, it should be positively treated by a Vim thalamotomy. (+info)
The IIANtr (PtsN) protein of Pseudomonas putida mediates the C source inhibition of the sigma54-dependent Pu promoter of the TOL plasmid.
The gene cluster adjacent to the sequence of rpoN (encoding sigma factor sigma54) of Pseudomonas putida has been studied with respect to the C source regulation of the Pu promoter of the upper TOL (toluene catabolism) operon. The region includes four open reading frames (ORFs), two of which (named ptsN and ptsO genes) encode proteins similar to components of the phosphoenolpyruvate:sugar phosphotransferase system. Each of the four genes was disrupted with a nonpolar insertion, and the effects in the inhibition caused by glucose on Pu activity were inspected with a lacZ reporter system. Although cells lacking ORF102, ORF284, and ptsO did not display any evident phenotype under the conditions tested, the loss of ptsN, which encodes the IIANtr protein, made Pu unresponsive to repression by glucose. The ptsN mutant had rates of glucose/gluconate consumption identical to those of the wild type, thus ruling out indirect effects mediated by the transport of the carbohydrate. A site-directed ptsN mutant in which the conserved phospho-acceptor site His68 of IIANtr was replaced by an aspartic acid residue made Pu blind to the presence or absence of glucose, thus supporting the notion that phosphorylation of IIANtr mediates the C source inhibition of the promoter. These data substantiate the existence of a molecular pathway for co-regulation of some sigma54 promoters in which IIANtr is a key protein intermediate. (+info)
Effect of organic solvents on the yield of solvent-tolerant Pseudomonas putida S12.
Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h-1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells. (+info)