Patients with severe slipped capital femoral epiphysis treated by the modified Dunn procedure have low rates of avascular necrosis, good outcomes, and little osteoarthritis at long-term follow-up.

AIMS: The modified Dunn procedure has the potential to restore the anatomy in hips with severe slipped capital femoral epiphyses (SCFE). However, there is a risk of developing avascular necrosis of the femoral head (AVN). In this paper, we report on clinical outcome, radiological outcome, AVN rate and complications, and the cumulative survivorship at long-term follow-up in patients undergoing the modified Dunn procedure for severe SCFE. PATIENTS AND METHODS: We performed a retrospective analysis involving 46 hips in 46 patients treated with a modified Dunn procedure for severe SCFE (slip angle > 60°) between 1999 and 2016. At nine-year-follow-up, 40 hips were available for clinical and radiological examination. Mean preoperative age was 13 years, and 14 hips (30%) presented with unstable slips. Mean preoperative slip angle was 64°. Kaplan-Meier survivorship was calculated. RESULTS: At the latest follow-up, the mean Merle d'Aubigné and Postel score was 17 points (14 to 18), mean modified Harris Hip Score was 94 points (66 to 100), and mean Hip Disability and Osteoarthritis Outcome Score was 91 points (67 to 100). Postoperative slip angle was 7° (1° to 16°). One hip (2%) had progression of osteoarthritis (OA). Two hips (5%) developed AVN of the femoral head and required further surgery. Three other hips (7%) underwent implant revision due to screw breakage or change of wires. Cumulative survivorship was 86% at ten-year follow-up. CONCLUSION: The modified Dunn procedure for severe SCFE resulted in a low rate of AVN, low risk of progression to OA, and high functional scores at long-term follow-up. The slip deformities were mainly corrected but secondary impingement deformities can develop in some hips and may require further surgical treatment. Cite this article: Bone Joint J 2019;101-B:403-414.

Indication of spatially random occurrence of Chlamydia-like organisms in Bufo bufo tadpoles from ponds located in the Geneva metropolitan area.

Occurrence of bacteria belonging to the order Chlamydiales was investigated for the first time in common toad (Bufo bufo) tadpole populations collected from 41 ponds in the Geneva metropolitan area, Switzerland. A Chlamydiales-specific real-time PCR was used to detect and amplify the Chlamydiales 16S ribosomal RNA-encoding gene from the tails of 375 tadpoles. We found the studied amphibian populations to host Chlamydia-like organisms (CLOs) attributable to the genera Similichlamydia, Neochlamydia, Protochlamydia and Parachlamydia (all belonging to the family Parachlamydiaceae), Simkania (family Simkaniaceae) and Estrella (family Criblamydiaceae); additionally, DNA from the genus Thermoanaerobacter (family Thermoanaerobacteriaceae) was detected. Global autocorrelation analysis did not reveal a spatial structure in the observed CLOs occurrence rates, and association tests involving land cover characteristics did not evidence any clear effect on CLOs occurrence rates in B. bufo. Although preliminary, these results suggest a random and ubiquitous distribution of CLOs in the environment, which would support the biogeographical expectation 'everything is everywhere' for the concerned microorganisms.

Dichloromethane biodegradation in multi-contaminated groundwater: Insights from biomolecular and compound-specific isotope analyses.

Dichloromethane (DCM) is a widespread and toxic industrial solvent which often co-occurs with chlorinated ethenes at polluted sites. Biodegradation of DCM occurs under both oxic and anoxic conditions in soils and aquifers. Here we investigated in situ and ex situ biodegradation of DCM in groundwater sampled from the industrial site of Themeroil (France), where DCM occurs as a major co-contaminant of chloroethenes. Carbon isotopic fractionation (εC) for DCM ranging from -46 to -22‰ were obtained under oxic or denitrifying conditions, in mineral medium or contaminated groundwater, and for laboratory cultures of Hyphomicrobium sp. strain GJ21 and two new DCM-degrading strains isolated from the contaminated groundwater. The extent of DCM biodegradation (B%) in the aquifer, as evaluated by compound-specific isotope analysis (δ13C), ranged from 1% to 85% applying DCM-specific εC derived from reference strains and those determined in this study. Laboratory groundwater microcosms under oxic conditions showed DCM biodegradation rates of up to 0.1 mM·day-1, with concomitant chloride release. Dehalogenase genes dcmA and dhlA involved in DCM biodegradation ranged from below 4 × 102 (boundary) to 1 × 107 (source zone) copies L-1 across the contamination plume. High-throughput sequencing on the 16S rrnA gene in groundwater samples showed that both contaminant level and terminal electron acceptor processes (TEAPs) influenced the distribution of genus-level taxa associated with DCM biodegradation. Taken together, our results demonstrate the potential of DCM biodegradation in multi-contaminated groundwater. This integrative approach may be applied to contaminated aquifers in the future, in order to identify microbial taxa and pathways associated with DCM biodegradation in relation to redox conditions and co-contamination levels.

On the importance of skewed offspring distributions and background selection in virus population genetics.

Many features of virus populations make them excellent candidates for population genetic study, including a very high rate of mutation, high levels of nucleotide diversity, exceptionally large census population sizes, and frequent positive selection. However, these attributes also mean that special care must be taken in population genetic inference. For example, highly skewed offspring distributions, frequent and severe population bottleneck events associated with infection and compartmentalization, and strong purifying selection all affect the distribution of genetic variation but are often not taken into account. Here, we draw particular attention to multiple-merger coalescent events and background selection, discuss potential misinference associated with these processes, and highlight potential avenues for better incorporating them into future population genetic analyses.

Bacterial communities in batch and continuous-flow wetlands treating the herbicide S-metolachlor.

Knowledge of wetland bacterial communities in the context of pesticide contamination and hydrological regime is scarce. We investigated the bacterial composition in constructed wetlands receiving Mercantor Gold(®) contaminated water (960 g L(-1) of the herbicide S-metolachlor, >80% of the S-enantiomer) operated under continuous-flow or batch modes to evaluate the impact of the hydraulic regime. In the continuous-flow wetland, S-metolachlor mass removal was >40%, whereas in the batch wetland, almost complete removal of S-metolachlor (93-97%) was observed. Detection of ethanesulfonic and oxanilic acid degradation products further indicated S-metolachlor biodegradation in the two wetlands. The dominant bacterial populations were characterised by terminal restriction fragment length polymorphism (T-RFLP) and 454 pyrosequencing. The bacterial profiles evolved during the first 35 days of the experiment, starting from a composition similar to that of inlet water, with the use of nitrate and to a lesser extent sulphate and manganese as terminal electron acceptors for microbial metabolism. Proteobacteria were the most abundant phylum, with Beta-, Alpha- and Gammaproteobacteria representing 26%, 19% and 17% respectively of total bacterial abundance. Bacterial composition in wetland water changed gradually over time in continuous-flow wetland and more abruptly in the batch wetland. Differences in overall bacterial water structure in the two systems were modest but significant (p=0.008), and S-metolachlor, nitrate, and total inorganic carbon concentrations correlated with changes in the bacterial profiles. Together, the results highlight that bacterial composition profiles and their dynamics may be used as bioindicators of herbicide exposure and hydraulic disturbances in wetland systems.

Using compound-specific isotope analysis to assess the degradation of chloroacetanilide herbicides in lab-scale wetlands.

Compound-specific isotope analysis (CSIA) is a promising tool to study the environmental fate of a wide range of contaminants including pesticides. In this study, a novel CSIA method was developed to analyse the stable carbon isotope signatures of widely used chloroacetanilide herbicides. The developed method was applied in combination with herbicide concentration and hydrochemical analyses to investigate in situ biodegradation of metolachlor, acetochlor and alachlor during their transport in lab-scale wetlands. Two distinct redox zones were identified in the wetlands. Oxic conditions prevailed close to the inlet of the four wetlands (oxygen concentration of 212±24µM), and anoxic conditions (oxygen concentrations of 28±41µM) prevailed towards the outlet, where dissipation of herbicides mainly occurred. Removal of acetochlor and alachlor from inlet to outlet of wetlands was 56% and 51%, whereas metolachlor was more persistent (23% of load dissipation). CSIA of chloroacetanilides at the inlet and outlet of the wetlands revealed carbon isotope fractionation of alachlor (εbulk=-2.0±0.3‰) and acetochlor (εbulk=-3.4±0.5‰), indicating that biodegradation contributes to the dissipation of both herbicides. This study is a first step towards the application of CSIA to evaluate the transport and degradation of chloroacetanilide herbicides in the environment.

Highly efficient methane biocatalysis revealed in a methanotrophic bacterium.

Methane is an essential component of the global carbon cycle and one of the most powerful greenhouse gases, yet it is also a promising alternative source of carbon for the biological production of value-added chemicals. Aerobic methane-consuming bacteria (methanotrophs) represent a potential biological platform for methane-based biocatalysis. Here we use a multi-pronged systems-level approach to reassess the metabolic functions for methane utilization in a promising bacterial biocatalyst. We demonstrate that methane assimilation is coupled with a highly efficient pyrophosphate-mediated glycolytic pathway, which under oxygen limitation participates in a novel form of fermentation-based methanotrophy. This surprising discovery suggests a novel mode of methane utilization in oxygen-limited environments, and opens new opportunities for a modular approach towards producing a variety of excreted chemical products using methane as a feedstock.

Evolution of uni- and bifactorial sexual compatibility systems in fungi.

Mating systems, that is, whether organisms give rise to progeny by selfing, inbreeding or outcrossing, strongly affect important ecological and evolutionary processes. Large variations in mating systems exist in fungi, allowing the study of their origin and consequences. In fungi, sexual incompatibility is determined by molecular recognition mechanisms, controlled by a single mating-type locus in most unifactorial fungi. In Basidiomycete fungi, however, which include rusts, smuts and mushrooms, a system has evolved in which incompatibility is controlled by two unlinked loci. This bifactorial system probably evolved from a unifactorial system. Multiple independent transitions back to a unifactorial system occurred. It is still unclear what force drove evolution and maintenance of these contrasting inheritance patterns that determine mating compatibility. Here, we give an overview of the evolutionary factors that might have driven the evolution of bifactoriality from a unifactorial system and the transitions back to unifactoriality. Bifactoriality most likely evolved for selfing avoidance. Subsequently, multiallelism at mating-type loci evolved through negative frequency-dependent selection by increasing the chance to find a compatible mate. Unifactoriality then evolved back in some species, possibly because either selfing was favoured or for increasing the chance to find a compatible mate in species with few alleles. Owing to the existence of closely related unifactorial and bifactorial species and the increasing knowledge of the genetic systems of the different mechanisms, Basidiomycetes provide an excellent model for studying the different forces that shape breeding systems.

Evolution in heterogeneous populations: from migration models to fixation probabilities.

Although dispersal is recognized as a key issue in several fields of population biology (such as behavioral ecology, population genetics, metapopulation dynamics or evolutionary modeling), these disciplines focus on different aspects of the concept and often make different implicit assumptions regarding migration models. Using simulations, we investigate how such assumptions translate into effective gene flow and fixation probability of selected alleles. Assumptions regarding migration type (e.g. source-sink, resident pre-emption, or balanced dispersal) and patterns (e.g. stepping-stone versus island dispersal) have large impacts when demes differ in sizes or selective pressures. The effects of fragmentation, as well as the spatial localization of newly arising mutations, also strongly depend on migration type and patterns. Migration rate also matters: depending on the migration type, fixation probabilities at an intermediate migration rate may lie outside the range defined by the low- and high-migration limits when demes differ in sizes. Given the extreme sensitivity of fixation probability to characteristics of dispersal, we underline the importance of making explicit (and documenting empirically) the crucial ecological/ behavioral assumptions underlying migration models.

Invasion and fixation of sex-reversal genes.

We simulated a meta-population with random dispersal among demes but local mating within demes to investigate conditions under which a dominant female-determining gene W, with no individual selection advantage, can invade and become fixed in females, changing the population from male to female heterogamety. Starting with one mutant W in a single deme, the interaction of sex ratio selection and random genetic drift causes W to be fixed among females more often than a comparable neutral mutation with no influence on sex determination, even when YY males have slightly reduced viability. Meta-population structure and interdeme selection can also favour the fixation of W. The reverse transition from female to male heterogamety can also occur with higher probability than for a comparable neutral mutation. These results help to explain the involvement of sex-determining genes in the evolution of sex chromosomes and in sexual selection and speciation.

Design and validation of a partial-genome microarray for transcriptional profiling of the Bradyrhizobium japonicum symbiotic gene region.

The design and use of a pilot microarray for transcriptome analysis of the symbiotic, nitrogen-fixing Bradyrhizobium japonicum is reported here. The custom-synthesized chip (Affymetrix GeneChip) features 738 genes, more than half of which belong to a 400-kb chromosomal segment strongly associated with symbiosis-related functions. RNA was isolated following an optimized protocol from wild-type cells grown aerobically and microaerobically, and from cells of aerobically grown regR mutant and microaerobically grown nifA mutant. Comparative microarray analyses thus revealed genes that are transcribed in either a RegR- or a NifA-dependent manner plus genes whose expression depends on the cellular oxygen status. Several genes were newly identified as members of the RegR and NifA regulons, beyond genes, which had been known from previous work. A comprehensive transcription analysis was performed with one of the new RegR-controlled genes (id880). Expression levels determined by microarray analysis of selected NifA- and RegR-controlled genes corresponded well with quantitative real-time PCR data, demonstrating the high complementarity of microarray analysis to classical methods of gene expression analysis in B. japonicum. Nevertheless, several previously established members of the NifA regulon were not detected as transcribed genes by microarray analysis, confirming the potential pitfalls of this approach also observed by other authors. By and large, this pilot study has paved the way towards the genome-wide transcriptome analysis of the 9.1-Mb B. japonicum genome.

The elusive roles of bacterial glutathione S-transferases: new lessons from genomes.

Glutathione S-transferases constitute a large family of enzymes which catalyze the addition of glutathione to endogenous or xenobiotic, often toxic electrophilic chemicals. Eukaryotic glutathione S-transferases usually promote the inactivation, degradation or excretion of a wide range of compounds by formation of the corresponding glutathione conjugates. In bacteria, by contrast, the few glutathione S-transferases for which substrates are known, such as dichloromethane dehalogenase, 1,2-dichloroepoxyethane epoxidase and tetrachlorohydroquinone reductase, are catabolic enzymes with an essential role for growth on recalcitrant chemicals. Glutathione S-transferase genes have also been found in bacterial operons and gene clusters involved in the degradation of aromatic compounds. Information from bacterial genome sequencing projects now suggests that glutathione S-transferases are present in large numbers in proteobacteria. In particular, the genomes of three Pseudomonas species each include at least ten different glutathione S-transferase genes. Several of the corresponding proteins define new classes of the glutathione S-transferase family and may also have novel functions that remain to be elucidated.

Dehalogenation of dichloromethane by dichloromethane dehalogenase/glutathione S-transferase leads to formation of DNA adducts.

Formation of DNA adducts following conversion of dichloromethane by bacterial dichloromethane dehalogenase/glutathione S-transferase was demonstrated. Adducts included dichloromethane carbon and glutathione sulfur atoms. A reaction with DNA occurred preferentially at guanine bases. Increased DNA degradation in a polA mutant of Methylobacterium dichloromethanicum DM4 grown with dichloromethane confirmed the genotoxicity associated with dichloromethane degradation, suggesting an important role of DNA repair in the metabolism of halogenated, DNA-alkylating compounds by bacteria.

Conjugation of haloalkanes by bacterial and mammalian glutathione transferases: mono- and dihalomethanes.

A primary route of metabolism of dihalomethanes occurs via glutathione (GSH) transferase-catalyzed conjugation. Mammalian theta class GSH transferases and a group of bacterial dichloromethane dehalogenases are able to catalyze the hydrolytic dehalogenation of dihalomethanes via GSH conjugation and subsequent formation of HCHO. Dihalomethanes have been shown to induce revertants in Salmonella typhimurium TA 1535 expressing theta class GSH transferases. Two mammalian theta class GSH transferases (rat GST 5-5 and human GST T1) and the bacterial dehalogenase DM11 were compared in the in vitro conjugation of CH(3)Cl and using in vitro assays (HCHO formation) and the S. typhimurium mutagenesis assay with the dihalomethanes CH(2)Cl(2), CH(2)Br(2), CH(2)BrCl, CH(2)ICl, CH(2)I(2), and CH(2)ClF. GSTs 5-5 and T1 had similar characteristics and exhibited first-order rather than Michaelis-Menten kinetics for HCHO formation over the range of dihalomethane concentrations tested. In contrast, the DM11 enzyme displayed typical hyperbolic Michaelis-Menten kinetics for all of the compounds tested. A similar pattern was observed for the conjugation of CH(3)Cl. The reversion tests with S. typhimurium expressing DM11 or GST 5-5 showed a concentration-dependent increase in revertants for most of the dihalomethanes, and DM11 produced revertants at dihalomethane concentrations lower than GST 5-5. Collectively, the results indicate that rates of conversion of dihalomethanes to HCHO are not correlated with mutagenicity and that GSH conjugates are genotoxic. The results are compared with the conjugation and genotoxicity of haloethanes in the preceding paper in this issue [Wheeler, J. B., Stourman, N. V., Armstrong, R. N., and Guengerich, F. P. (2001) Chem. Res. Toxicol. 14, 1107-1117]. The halide order appears most important in the dihalomethane conjugation reactions catalyzed by GST 5-5 and less so in GST T1 and DM11, probably due to changes in the rate-limiting steps.

Dichloromethane mediated in vivo selection and functional characterization of rat glutathione S-transferase theta 1-1 variants.

Methylobacterium dichloromethanicum DM4 is able to grow with dichloromethane as the sole carbon and energy source by using a dichloromethane dehalogenase/glutathione S-transferase (GST) for the conversion of dichloromethane to formaldehyde. Mammalian homologs of this bacterial enzyme are also known to catalyze this reaction. However, the dehalogenation of dichloromethane by GST T1-1 from rat was highly mutagenic and toxic to methylotrophic bacteria. Plasmid-driven expression of rat GST T1-1 in strain DM4-2cr, a mutant of strain DM4 lacking dichloromethane dehalogenase, reduced cell viability 10(5)-fold in the presence of dichloromethane. This effect was exploited to select dichloromethane-resistant transconjugants of strain DM4-2cr carrying a plasmid-encoded rGSTT1 gene. Transconjugants that still expressed the GST T1 protein after dichloromethane treatment included rGSTT1 mutants encoding protein variants with sequence changes from the wild-type ranging from single residue exchanges to large insertions and deletions. A structural model of rat GST T1-1 suggested that sequence variation was clustered around the glutathione activation site and at the protein C-terminus believed to cap the active site. The enzymatic activity of purified His-tagged GST T1-1 variants expressed in Escherichia coli was markedly reduced with both dichloromethane and the alternative substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane. These results provide the first experimental evidence for the involvement of Gln102 and Arg107 in catalysis, and illustrate the potential of in vivo approaches to identify catalytic residues in GSTs whose activity leads to toxic effects.

Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4.

The cmuA and cmuB genes are required for growth of Methylobacterium chloromethanicum strain CM4 with chloromethane as the sole carbon source. While CmuB was previously shown to possess methylcobalamin:tetrahydrofolate methyltransferase activity, sequence analysis indicated that CmuA represented a novel and so far unique two-domain methyltransferase/corrinoid-binding protein involved in methyl transfer from chloromethane to a corrin moiety. CmuA was purified from wild-type M. chloromethanicum strain CM4 and characterized as a monomeric, cobalt-containing and zinc-containing enzyme of molecular mass 67 kDa with a bound vitamin B12 cofactor. In combination, CmuA and CmuB proteins catalyze the in vitro transfer of the methyl group of chloromethane to tetrahydrofolate, thus affording a direct link between chloromethane dehalogenation and core C1 metabolism of Methylobacterium. Chloromethane dehalogenase activity in vitro is limited by CmuB, as formation of methyltetrahydrofolate from chloromethane displays apparent Michaelis-Menten kinetics with respect to methylated CmuA, with an apparent Km of 0.27 microM and a Vmax of 0.45 U x mg(-1). This contrasts with sequence-related systems for methyl transfer from methanogens, which involve methyltransferase and corrinoid protein components in well-defined stoichiometric ratios.

Chloromethane utilization gene cluster from Hyphomicrobium chloromethanicum strain CM2(T) and development of functional gene probes to detect halomethane-degrading bacteria.

Hyphomicrobium chloromethanicum CM2(T), an aerobic methylotrophic member of the alpha subclass of the class proteobacteria, can grow with chloromethane as the sole carbon and energy source. H. chloromethanicum possesses an inducible enzyme system for utilization of chloromethane, in which two polypeptides (67-kDa CmuA and 35-kDa CmuB) are expressed. Previously, four genes, cmuA, cmuB, cmuC, and purU, were shown to be essential for growth of Methylobacterium chloromethanicum on chloromethane. The cmuA and cmuB genes were used as probes to identify homologs in H. chloromethanicum. A cmu gene cluster (9.5 kb) in H. chloromethanicum contained 10 open reading frames: folD (partial), pduX, orf153, orf207, orf225, cmuB, cmuC, cmuA, fmdB, and paaE (partial). CmuA from H. chloromethanicum (67 kDa) showed high identity to CmuA from M. chloromethanicum and contains an N-terminal methyltransferase domain and a C-terminal corrinoid-binding domain. CmuB from H. chloromethanicum is related to a family of methyl transfer proteins and to the CmuB methyltransferase from M. chloromethanicum. CmuC from H. chloromethanicum shows identity to CmuC from M. chloromethanicum and is a putative methyltransferase. folD codes for a methylene-tetrahydrofolate cyclohydrolase, which may be involved in the C(1) transfer pathway for carbon assimilation and CO(2) production, and paaE codes for a putative redox active protein. Molecular analyses and some preliminary biochemical data indicated that the chloromethane utilization pathway in H. chloromethanicum is similar to the corrinoid-dependent methyl transfer system in M. chloromethanicum. PCR primers were developed for successful amplification of cmuA genes from newly isolated chloromethane utilizers and enrichment cultures.

DNA polymerase I is essential for growth of Methylobacterium dichloromethanicum DM4 with dichloromethane.

Methylobacterium dichloromethanicum DM4 grows with dichloromethane as the unique carbon and energy source by virtue of a single enzyme, dichloromethane dehalogenase-glutathione S-transferase. A mutant of the dichloromethane-degrading strain M. dichloromethanicum DM4, strain DM4-1445, was obtained by mini-Tn5 transposon mutagenesis that was no longer able to grow with dichloromethane. Dichloromethane dehalogenase activity in this mutant was comparable to that of the wild-type strain. The site of mini-Tn5 insertion in this mutant was located in the polA gene encoding DNA polymerase I, an enzyme with a well-known role in DNA repair. DNA polymerase activity was not detected in cell extracts of the polA mutant. Conjugation of a plasmid containing the intact DNA polymerase I gene into the polA mutant restored growth with dichloromethane, indicating that the polA gene defect was responsible for the observed lack of growth of this mutant with dichloromethane. Viability of the DM4-1445 mutant was strongly reduced upon exposure to both UV light and dichloromethane. The polA'-lacZ transcriptional fusion resulting from mini-Tn5 insertion was constitutively expressed at high levels and induced about twofold after addition of 10 mM dichloromethane. Taken together, these data indicate that DNA polymerase I is essential for growth of M. dichloromethanicum DM4 with dichloromethane and further suggest an important role of the DNA repair machinery in the degradation of halogenated, DNA-alkylating compounds by bacteria.

Properties of the methylcobalamin:H4folate methyltransferase involved in chloromethane utilization by Methylobacterium sp. strain CM4.

Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA 96, 4615-4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol x min-1 x (mg protein)-1. The apparent kinetic constants for H4folate were: Km = 240 microM, Vmax = 28.5 nmol x min-1 x (mg protein)-1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mM, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.

Enzyme-mediated dichloromethane toxicity and mutagenicity of bacterial and mammalian dichloromethane-active glutathione S-transferases.

The kinetic properties of bacterial and rat liver glutathione S-transferases (GST) active with dichloromethane (DCM) were compared. The theta class glutathione S-transferase (rGSTTI-1) from rat liver had an affinity for dihalomethanes lower by three orders of magnitude (K(app) > 50 mM) than the bacterial DCM dehalogenase/GST from Methylophilus sp. DM11. Unlike the bacterial DCM dehalogenase, the rat enzyme was unable to support growth of the dehalogenase minus Methylobacterium sp. DM4-2cr mutant with DCM. Moreover, the presence of DCM inhibited growth with methanol of the DM4-2cr transconjugant expressing the rat liver GSTT1-1. In Salmonella typhimurium TA1535, expression of rat and bacterial DCM-active GST from a plasmid in the presence of DCM yielded up to 5.3 times more reversions to histidine prototrophy in the transconjugant expressing the rat enzyme. Under the same conditions, however, GST-mediated conversion of DCM to formaldehyde was lower in cell-free extracts of the transconjugant expressing the rat GSTT1 than in the corresponding strain expressing the bacterial DCM dehalogenase. This provided new evidence that formaldehyde was not the main toxicant associated with GST-mediated DCM conversion, and indicated that an intermediate in the transformation of DCM by GST, presumably S-chloromethylglutathione, was responsible for the observed effects. The marked differences in substrate affinity of rat and bacterial DCM-active GST, as well as in the toxicity and genotoxicity associated with expression of these enzymes in bacteria, suggest that bacterial DCM dehalogenases/GST have evolved to minimise the toxic effects associated with glutathione-mediated catalysis of DCM conversion.