[Intestinal microbiota in individualized therapies]. / Die intestinale Mikrobiota als Ansatz für individuelle Therapien.

During recent years, the analysis of the human microbiota has been receiving more and more scientific focus. Deep sequencing analysis enables characterization of microbial communities in different environments without the need of culture-based methods. Hereby, information about microbial communities is increasing enormously. Numerous studies in humans and animal models revealed the important role of the microbiome in emergence and natural course of diseases such as autoimmune diseases and metabolic disorders, e. g., the metabolic syndrome. The identification of causalities between the intestinal microbiota composition and function, and diseases in humans and animal models can help to develop individualized therapies targeting the microbiome and its modification. Nowadays, it is established that several factors influence the composition of the microbiota. Diet it is one of the major factors shaping the microbiota and the use of pro- and prebiotica may induce changes in the microbial community. Fecal microbiome transfer is the first approach targeting the intestinal microbiota which is implemented in the clinical routine for patients with therapy-refractory infections with Clostridium difficile. Herewith, the recipient's microbiota can be changed permanently and the patient can be cured from the infection.

In vitro colonisation of the distal colon by Akkermansia muciniphila is largely mucin and pH dependent.

Host mucin is the main constituent of the mucus layer that covers the gut epithelium of the host, and an important source of glycans for the bacteria colonising the intestine. Akkermansia muciniphila is a mucin-degrading bacterium, abundant in the human gut, that is able to produce acetate and propionate during this degradation process. A. muciniphila has been correlated with human health in previous studies, but a mechanistic explanation is lacking. In this study, the main site of colonisation was characterised alongside additional conditions, such as differences in colon pH, prebiotic supplementation and variable mucin supply. To overcome the limitations of in vivo studies concerning variations in mucin availability and difficult access to proximal regions of the colon, a dynamic in vitro gut model (SHIME) was used. In this model, A. muciniphila was found to colonise the distal colon compartment more abundantly than the proximal colon ((±8 log copies/ml compared to ±4 log copies/ml) and the preference for the distal compartment was found to be pH-dependent. The addition of mucin caused a specific increase of A. muciniphila (±4.5 log increase over two days), far exceeding the response of other bacteria present, together with an increase in propionate. These findings suggest that colonisation and mucin degradation by A. muciniphila is dependent on pH and the concentration of mucin. Our results revealed the preference of A. muciniphila for the distal colon environment due to its higher pH and uncovered the quick and stable response of A. muciniphila to mucin supplementation.

Microbial Toluene Removal in Hypoxic Model Constructed Wetlands Occurs Predominantly via the Ring Monooxygenation Pathway.

In the present study, microbial toluene degradation in controlled constructed wetland model systems, planted fixed-bed reactors (PFRs), was queried with DNA-based methods in combination with stable isotope fractionation analysis and characterization of toluene-degrading microbial isolates. Two PFR replicates were operated with toluene as the sole external carbon and electron source for 2 years. The bulk redox conditions in these systems were hypoxic to anoxic. The autochthonous bacterial communities, as analyzed by Illumina sequencing of 16S rRNA gene amplicons, were mainly comprised of the families Xanthomonadaceae, Comamonadaceae, and Burkholderiaceae, plus Rhodospirillaceae in one of the PFR replicates. DNA microarray analyses of the catabolic potentials for aromatic compound degradation suggested the presence of the ring monooxygenation pathway in both systems, as well as the anaerobic toluene pathway in the PFR replicate with a high abundance of Rhodospirillaceae. The presence of catabolic genes encoding the ring monooxygenation pathway was verified by quantitative PCR analysis, utilizing the obtained toluene-degrading isolates as references. Stable isotope fractionation analysis showed low-level of carbon fractionation and only minimal hydrogen fractionation in both PFRs, which matches the fractionation signatures of monooxygenation and dioxygenation. In combination with the results of the DNA-based analyses, this suggests that toluene degradation occurs predominantly via ring monooxygenation in the PFRs.

Quantitative response of nitrifying and denitrifying communities to environmental variables in a full-scale membrane bioreactor.

The abundance and transcription levels of specific gene markers of total bacteria, ammonia-oxidizing Betaproteobacteria, nitrite-oxidizing bacteria (Nitrospira-like) and denitrifiers (N2O-reducers) were analyzed using quantitative PCR (qPCR) and reverse-transcription qPCR during 9 months in a full-scale membrane bioreactor treating urban wastewater. A stable community of N-removal key players was developed; however, the abundance of active populations experienced sharper shifts, demonstrating their fast adaptation to changing conditions. Despite constituting a small percentage of the total bacterial community, the larger abundances of active populations of nitrifiers explained the high N-removal accomplished by the MBR. Multivariate analyses revealed that temperature, accumulation of volatile suspended solids in the sludge, BOD5, NH4(+) concentration and C/N ratio of the wastewater contributed significantly (23-38%) to explain changes in the abundance of nitrifiers and denitrifiers. However, each targeted group showed different responses to shifts in these parameters, evidencing the complexity of the balance among them for successful biological N-removal.

Prevalence and molecular epidemiology of meticillin-resistant Staphylococcus aureus in nursing home residents in northern Germany.

Nursing home residents are a population at risk for carrying meticillin-resistant Staphylococcus aureus (MRSA). To better guide infection control and healthcare network initiatives, we investigated the point prevalence and molecular epidemiology of MRSA colonisation among nursing home residents in Brunswick, northern Germany. Among the 32 participating nursing homes of the available 34 in the region, 68% of residents (1827 of 2688) were screened for nasal and/or wound colonisation. A total of 139 residents (7.6%; 95% confidence interval: 6.4-8.8%) were identified as MRSA positive, almost six-fold more than the 24 MRSA carriers (0.9%) expected according to the nursing homes' pre-test information. Although known risk factors including urinary tract catheters, wounds, preceding hospital admission, and high grade resident care were confirmed, none was sensitive enough to be considered as the sole determinant of MRSA carriage. spa typing revealed that more than 70% of isolates belonged to the Barnim strain (ST-22, EMRSA-15, CC22) typical for hospital-acquired MRSA in northern Germany. There was no evidence for the presence of community-acquired or livestock-associated S. aureus strains. These data show that in northern Germany MRSA has spread from the hospital environment to other healthcare institutions, which must now be regarded as important reservoirs for MRSA transmission.

Chlorophenol hydroxylases encoded by plasmid pJP4 differentially contribute to chlorophenoxyacetic acid degradation.

Phenoxyalkanoic compounds are used worldwide as herbicides. Cupriavidus necator JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using tfd functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the tfdB(I) and tfdB(II) genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the tfdB(I) and tfdB(II) genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdB(I) contributes to a significantly higher extent than TfdB(II). Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the tfdB genes enhanced the toxic effect of 2,4-DCP on C. necator cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by C. necator JMP134 and by a different host, Burkholderia xenovorans LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two tfdB genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds.

Efficient turnover of chlorocatechols is essential for growth of Ralstonia eutropha JMP134(pJP4) in 3-chlorobenzoic acid.

Ralstonia eutropha JMP134(pJP4) degrades 3-chlorobenzoate (3-CB) by using two not completely isofunctional, pJP4-encoded chlorocatechol degradation gene clusters, tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II). Introduction of several copies of each gene cluster into R. eutropha JMP222, which lacks pJP4 and thus accumulates chlorocatechols from 3-CB, allows the derivatives to grow in this substrate. However, JMP222 derivatives containing one chromosomal copy of each cluster did not grow in 3-CB. The failure to grow in 3-CB was the result of accumulation of chlorocatechols due to the limiting activity of chlorocatechol 1,2-dioxygenase (TfdC), the first enzyme in the chlorocatechol degradation pathway. Micromolar concentrations of 3- and 4-chlorocatechol inhibited the growth of strains JMP134 and JMP222 in benzoate, and cells of strain JMP222 exposed to 3 mM 3-CB exhibited a 2-order-of-magnitude decrease in viability. This toxicity effect was not observed with strain JMP222 harboring multiple copies of the tfdC(I) gene, and the derivative of strain JMP222 containing tfdC(I)D(I)E(I)F(I) plus multiple copies of the tfdC(I) gene could efficiently grow in 3-CB. In addition, tfdC(I) and tfdC(II) gene mutants of strain JMP134 exhibited no growth and impaired growth in 3-CB, respectively. The introduction into strain JMP134 of the xylS-xylXYZL genes, encoding a broad-substrate-range benzoate 1,2-dioxygenase system and thus increasing the transformation of 3-CB into chlorocatechols, resulted in derivatives that exhibited a sharp decrease in the ability to grow in 3-CB. These observations indicate that the dosage of chlorocatechol-transforming genes is critical for growth in 3-CB. This effect depends on a delicate balance between chlorocatechol-producing and chlorocatechol-consuming reactions.

Bacterial community dynamics during biostimulation and bioaugmentation experiments aiming at chlorobenzene degradation in groundwater.

A set of microcosm experiments was performed to assess different bioremediation strategies, i.e., biostimulation and bioaugmentation, for groundwater contaminated with chlorobenzenes. The biodegradative potential was stimulated either by the supply of electron acceptors (air, (NO3-), to increase the activity of the indigenous bacterial community, or by the addition of aerobic chlorobenzene-degrading bacteria (Pseudomonas putida GJ31, Pseudomonas aeruginosa RHO1, Pseudomonas putida F1deltaCC). Experiments were performed with natural groundwater of the aquifer of Bitterfeld, which had been contaminated with 1,2-dichlorobenzene (1,2-DCB), 1,4-dichlorobenzene (1,4-DCB), and chlorobenzene (CB). The microcosms consisted of airtight glass bottles with 800 mL of natural groundwater and were incubated under in situ temperature (13 degrees C). Behavior of the introduced strains within the indigenous bacterial community was monitored by fluorescent in situ hybridization (FISH) with species-specific oligonucleotides. Dynamics of the indigenous community and the introduced strains within the microcosms were followed by single-strand conformation polymorphism (SSCP) analysis of 16S rDNA amplicons obtained from total DNA of the microbial community. An indigenous biodegradation potential under aerobic as well as anaerobic denitrifying conditions was observed accompanied by fast and specific changes in the natural bacterial community composition. Augmentation with P. aeruginosa RHO1 did not enhance bio-degradation. In contrast, both P. putida GJ31 as well as P. putida F1deltaCC were capable of growing in groundwater, even in the presence of the natural microbial community, and thereby stimulating chlorobenzene depletion. P. putida GJ31 disappeared when the xenobiotics were depleted and P. putida F1deltaCC persisted even in the absence of CB. Detailed statistical analyses revealed that community dynamics of the groundwater microbiota were highly reproducible but specific to the introduced strain, its inoculum size, and the imposed physicochemical conditions. These findings could contribute to the design of better in situ bioremediation strategies for contaminated groundwater.

Transformation of chlorinated benzenes and toluenes by Ralstonia sp. strain PS12 tecA (tetrachlorobenzene dioxygenase) and tecB (chlorobenzene dihydrodiol dehydrogenase) gene products.

The tecB gene, located downstream of tecA and encoding tetrachlorobenzene dioxygenase, in Ralstonia sp. strain PS12 was cloned into Escherichia coli DH5alpha together with the tecA gene. The identity of the tecB gene product as a chlorobenzene dihydrodiol dehydrogenase was verified by transformation into the respective catechols of chlorobenzene, the three isomeric dichlorobenzenes, as well as 1,2,3- and 1,2,4-trichlorobenzenes, all of which are transformed by TecA into the respective dihydrodihydroxy derivatives. Di- and trichlorotoluenes were either subject to TecA-mediated dioxygenation (the major or sole reaction observed for the 1,2,4-substituted 2,4-, 2,5-, and 3,4-dichlorotoluenes), resulting in the formation of the dihydrodihydroxy derivatives, or to monooxygenation of the methyl substituent (the major or sole reaction observed for 2,3-, 2,6-, and 3,5-dichloro- and 2,4,5-trichlorotoluenes), resulting in formation of the respective benzyl alcohols. All of the chlorotoluenes subject to dioxygenation by TecA were transformed, without intermediate accumulation of dihydrodihydroxy derivatives, into the respective catechols by TecAB, indicating that dehydrogenation is no bottleneck for chlorobenzene or chlorotoluene degradation. However, only those chlorotoluenes subject to a predominant dioxygenation were growth substrates for PS12, confirming that monooxygenation is an unproductive pathway in PS12.

Engineering bacteria for bioremediation.

The treatment of environmental pollution by microorganisms is a promising technology. Various genetic approaches have been developed and used to optimize the enzymes, metabolic pathways and organisms relevant for biodegradation. New information on the metabolic routes and bottlenecks of degradation is still accumulating, enlarging the available toolbox. With molecular methods allowing the characterization of microbial community structure and activities, the performance of microorganisms under in situ conditions and in concert with the indigenous microflora will become predictable.

Role of tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II) gene modules in catabolism of 3-chlorobenzoate by Ralstonia eutropha JMP134(pJP4).

The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(II)F(II) (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR-tfdC(I)D(I)E(I)F(I) grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD(II)C(II)E(II)F(II). In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.

Bacteria designed for bioremediation.

Although many environmental pollutants are efficiently degraded by microorganisms, others persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.

NahW, a novel, inducible salicylate hydroxylase involved in mineralization of naphthalene by Pseudomonas stutzeri AN10.

Two genes, nahG and nahW, encoding two independent salicylate 1-hydroxylases have been identified in the naphthalene-degrading strain Pseudomonas stutzeri AN10. While nahG resides in the same transcriptional unit as the meta-cleavage pathway genes, forming the naphthalene degradation lower pathway, nahW is situated outside but in close proximity to this transcriptional unit. The nahG and nahW genes of P. stutzeri AN10 are induced and expressed upon incubation with salicylate, and the enzymes that are encoded, NahG and NahW, are involved in naphthalene and salicylate metabolism. Both genes, nahG and nahW, have been cloned in Escherichia coli JM109. The overexpression of these genes yields peptides with apparent molecular masses of 46 kDa (NahG) and 43 kDa (NahW), respectively. Both enzymes exhibit broad substrate specificities and metabolize salicylate, methylsalicylates, and chlorosalicylates. However, the relative rates by which the substituted analogs are transformed differ considerably.

Initial reactions in the biodegradation of 1-chloro-4-nitrobenzene by a newly isolated bacterium, strain LW1.

Bacterial strain LW1, which belongs to the family Comamonadaceae, utilizes 1-chloro-4-nitrobenzene (1C4NB) as a sole source of carbon, nitrogen, and energy. Suspensions of 1C4NB-grown cells removed 1C4NB from culture fluids, and there was a concomitant release of ammonia and chloride. Under anaerobic conditions LW1 transformed 1C4NB into a product which was identified as 2-amino-5-chlorophenol by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. This transformation indicated that there was partial reduction of the nitro group to the hydroxylamino substituent, followed by Bamberger rearrangement. In the presence of oxygen but in the absence of NAD, fast transformation of 2-amino-5-chlorophenol into a transiently stable yellow product was observed with resting cells and cell extracts. This compound exhibited an absorption maximum at 395 nm and was further converted to a dead-end product with maxima at 226 and 272 nm. The compound formed was subsequently identified by 1H and 13C NMR spectroscopy and mass spectrometry as 5-chloropicolinic acid. In contrast, when NAD was added in the presence of oxygen, only minor amounts of 5-chloropicolinic acid were formed, and a new product, which exhibited an absorption maximum at 306 nm, accumulated.

Genetic and biochemical analyses of the tec operon suggest a route for evolution of chlorobenzene degradation genes.

The TecA broad-spectrum chlorobenzene dioxygenase of Burkholderia sp. strain PS12 catalyzes the first step in the mineralization of 1,2,4, 5-tetrachlorobenzene. The catabolic genes were localized on a small plasmid that belongs to the IncPbeta incompatibility group. PCR analysis of the genetic environment of the tec genes indicated high similarity to the transposon-organized catabolic tcb chlorobenzene degradation genes of Pseudomonas sp. strain P51. Sequence analysis of the regions flanking the tecA genes revealed an upstream open reading frame (ORF) with high similarity to the todF 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase gene of Pseudomonas putida F1 and a discontinuous downstream ORF showing high similarity to the todE catechol 2,3-dioxygenase gene of strain F1. Both homologues in strain P51 exist only as deletion remnants. We suggest that different genetic events thus led to inactivation of the perturbing meta-cleavage enzymes in strains P51 and PS12 during the evolution of efficient chlorobenzene degradation pathways. Biochemical characterization of TodF-like protein TlpF and a genetically refunctionalized TodE-like protein, TlpE, produced in Escherichia coli provided data consistent with the proposed relationships.

Identification of chlorobenzene dioxygenase sequence elements involved in dechlorination of 1,2,4,5-tetrachlorobenzene.

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate specificities. Escherichia coli cells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene. A hybrid TecA dioxygenase variant containing the large alpha-subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity. Acquisition of dehalogenase activity was achieved by replacement of specific todC1 alpha-subunit subsequences by equivalent sequences of the tecA1 alpha-subunit. Substrate transformation specificities and rates by E. coli resting cells expressing hybrid systems were analyzed by high-performance liquid chromatography. This allowed the identification of both a single amino acid and potentially interacting regions required for dechlorination of tetrachlorobenzene. Hybrids with extended substrate ranges were generated that exhibited activity toward both benzene and tetrachlorobenzene. The regions determining substrate specificity in (chloro)benzene dioxygenases appear to be different from those previously identified in biphenyl dioxygenases.

Biochemical and genetic characterization of a gentisate 1, 2-dioxygenase from Sphingomonas sp. strain RW5.

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488-6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (Km) and specificity constants (Kcat/Km) of the enzyme were determined to be 15 microM and 511 s-1 M-1 x 10(4) for gentisate and 754 microM and 20 s-1 M-1 x 10(4) for 3, 6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.

Characterization of a gene cluster from Ralstonia eutropha JMP134 encoding metabolism of 4-methylmuconolactone.

A 2,585 bp chromosomal DNA segment of Ralstonia eutropha JMP134 (formerly: Alcaligenes eutrophus JMP134) which contains a gene cluster encoding part of the modified ortho-cleavage pathway encodes a putative transport protein for 4-methylmuconolactone, a novel 4-methylmuconolactone methylisomerase and methylmuconolactone isomerase. The putative 4-methylmuconolactone transporter, a protein with a calculated molecular mass of 45.8 kDa, exhibits sequence homology to other members of the major superfamily of transmembrane facilitators and shows the common structural motif of 12 transmembrane-spanning alpha-helical segments and the hallmark amino acid motif characteristic of the superfamily. Consistent with the novelty of the reaction catalyzed by 4-methylmuconolactone methylisomerase, no primary sequence homologies were found between this enzyme or its gene and other proteins or genes in the data banks, suggesting that this enzyme represents a new type of isomerase. The molecular mass of the native 4-methylmuconolactone methylisomerase was determined by gel filtration analysis to be 25 +/- 2 kDa. From the polynucleotide sequence of the gene, a molecular mass of 12.9 kDa was calculated and hence we predict a homodimeric quaternary structure. The high sensitivity of 4-methylmuconolactone methylisomerase to heavy metals and thiol-modifying reagents implicates the involvement of sulfhydryl groups in the catalytic reaction. The methylmuconolactone isomerase - calculated molecular mass 10.3 kDa - has a primary structure related to the classical muconolactone isomerases (EC 5.3.3.4) of Acinetobacter calcoaceticus, of two Pseudomonas putida strains and of Ralstonia eutropha JMP134, suggesting that these are all isoenzymes. Consistent with this proposal is the finding that the purified protein exhibits muconolactone-isomerizing activity.

Detoxification of protoanemonin by dienelactone hydrolase.

Protoanemonin is a toxic metabolite which may be formed during the degradation of some chloroaromatic compounds, such as polychlorinated biphenyls, by natural microbial consortia. We show here that protoanemonin can be transformed by dienelactone hydrolase of Pseudomonas sp. strain B13 to cis-acetylacrylate. Although similar Km values were observed for cis-dienelactone and protoanemonin, the turnover rate of protoanemonin was only 1% that of cis-dienelactone. This indicates that at least this percentage of the enzyme is in the active state, even in the absence of activation. The trans-dienelactone hydrolase of Pseudomonas sp. strain RW10 did not detectably transform protoanemonin. Obviously, Pseudomonas sp. strain B13 possesses at least two mechanisms to avoid protoanemonin toxicity, namely a highly active chloromuconate cycloisomerase, which routes most of the 3-chloro-cis,cis-muconate to the cis-dienelactone, thereby largely preventing protoanemonin formation, and dienelactone hydrolase, which detoxifies any small amount of protoanemonin that might nevertheless be formed.

Evidence for an isomeric muconolactone isomerase involved in the metabolism of 4-methylmuconolactone by Alcaligenes eutrophus JMP134.

An enzyme specifically induced during 4-methylmuconolactone metabolism by Alcaligenes eutrophus JMP 134 and that exhibited muconolactone isomerizing activity was purified to homogeneity. The enzyme, involved in the isomerization of 3-methylmuconolactone had a high degree of sequence similarity with muconolactone isomerase of Alcaligenes eutrophus JMP 134 and other previously described muconolactone isomerases of the 3-oxoadipate pathway. Kinetic analysis showed that the enzyme has a substrate spectrum and a reaction mechanism similar to those of the muconolactone isomerase, but that it has distinct kinetic properties.