Stereo- and regiospecific cis,cis-muconate cycloisomerization by Rhodococcus rhodochrous N75

cis,cis-Muconate cycloisomerase was purified to homogeneity from cells of Rhodococcus rhodochrous N75 grown at the expense of benzoate and p-toluate as the sole sources of carbon. A single cycloisomerase was found to be induced in this organism with no isoforms being detected when R. rhodochrous N75 was grown on either benzoate or p-toluate as the sole source of carbon. The enzyme is hexameric with a single subunit Mr of 40,000. cis,cis-Muconate cycloisomerase from R. rhodochrous N75 displayed strict regio- and stereospecificity whereby cis,cis-muconate is cycloisomerized to (4S)-muconolactone and 2-methyl- and 3-methyl-substituted muconates are cycloisomerized to 2-methyl- and 4-methyl-substituted muconolactones by 1,4- and 3,6-cycloisomerization, respectively.     

Metabolism of dichloromethylcatechols as central intermediates in the degradation of dichlorotoluenes by Ralstonia sp. strain PS12

Ralstonia sp. strain PS12 is able to use 2,4-, 2,5-, and 3,4-dichlorotoluene as growth substrates. Dichloromethylcatechols are central intermediates that are formed by TecA tetrachlorobenzene dioxygenase-mediated activation at two adjacent unsubstituted carbon atoms followed by TecB chlorobenzene dihydrodiol dehydrogenase-catalyzed rearomatization and then are channeled into a chlorocatechol ortho cleavage pathway involving a chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and dienelactone hydrolase. However, completely different metabolic routes were observed for the three dichloromethylcatechols analyzed. Whereas 3,4-dichloro-6-methylcatechol is quantitatively transformed into one dienelactone (5-chloro-2-methyldienelactone) and thus is degraded via a linear pathway, 3,5-dichloro-2-methylmuconate formed from 4,6-dichloro-3-methylcatechol is subject to both 1,4- and 3,6-cycloisomerization and thus is degraded via a branched metabolic route. 3,6-Dichloro-4-methylcatechol, on the first view, is transformed predominantly into one (2-chloro-3-methyl-trans-) dienelactone. In situ (1)H nuclear magnetic resonance analysis revealed the intermediate formation of 2,5-dichloro-4-methylmuconolactone, showing that both 1,4- and 3,6-cycloisomerization occur with this muconate and indicating a degradation of the muconolactone via a reversible cycloisomerization reaction and the dienelactone-forming branch of the pathway. Diastereomeric mixtures of two dichloromethylmuconolactones were prepared chemically to proof such a hypothesis. Chloromuconate cycloisomerase transformed 3,5-dichloro-2-methylmuconolactone into a mixture of 2-chloro-5-methyl-cis- and 3-chloro-2-methyldienelactone, affording evidence for a metabolic route of 3,5-dichloro-2-methylmuconolactone via 3,5-dichloro-2-methylmuconate into 2-chloro-5-methyl-cis-dienelactone. 2,5-Dichloro-3-methylmuconolactone was transformed nearly exclusively into 2-chloro-3-methyl-trans-dienelactone.     

Purification and characterization of 4-methylmuconolactone methyl-isomerase, a novel enzyme of the modified 3-oxoadipate pathway in nocardioform actinomycetes.

The novel enzyme 4-methyl-2-enelactone methyl-isomerase was detected in, and purified to electrophoretic homogeneity from, p-toluate-grown cells of Rhodococcus rhodocrous N75, a nocardioform actinomycete. The enzyme was very thermostable and had a native Mr of 75,500; as the monomer had an Mr of 17,000, the enzyme is probably tetrameric. The new isomerase is highly specific with respect to its lactone substrate, only accepting (+)-(4S)-4-methylmuconolactone (4-carboxymethyl-4-methylbut-2-en-1,4-olide), and the putative isomerization reaction intermediate 1-methylbislactone ((-)-1-methyl-3,7-dioxo-2,6-dioxabicyclo-[3.3.0]octane) as substrates, and yielding (-)-(4S)-3-methylmuconolactone (4-carboxymethyl-3-methylbut-2-en-1,4-olide) as product. Some other lactone analogues acted as competitive inhibitors. Our data suggest that the isomerization does not involve actual methyl migration, but proceeds via the 1-methybislactone.     

Chloromethylmuconolactones as critical metabolites in the degradation of chloromethylcatechols: recalcitrance of 2-chlorotoluene

To elucidate possible reasons for the recalcitrance of 2-chlorotoluene, the metabolism of chloromethylcatechols, formed after dioxygenation and dehydrogenation by Ralstonia sp. strain PS12 tetrachlorobenzene dioxygenase and chlorobenzene dihydrodiol dehydrogenase, was monitored using chlorocatechol dioxygenases and chloromuconate cycloisomerases partly purified from Ralstonia sp. strain PS12 and Wautersia eutropha JMP134. Two chloromethylcatechols, 3-chloro-4-methylcatechol and 4-chloro-3-methylcatechol, were formed from 2-chlorotoluene. 3-Chloro-4-methylcatechol was transformed into 5-chloro-4-methylmuconolactone and 2-chloro-3-methylmuconolactone. For mechanistic reasons neither of these cycloisomerization products can be dehalogenated by chloromuconate cycloisomerases, with the result that 3-chloro-4-methylcatechol cannot be mineralized by reaction sequences related to catechol ortho-cleavage pathways known thus far. 4-Chloro-3-methylcatechol is only poorly dehalogenated during enzymatic processing due to the kinetic properties of the chloromuconate cycloisomerases. Thus, degradation of 2-chlorotoluene via a dioxygenolytic pathway is evidently problematic. In contrast, 5-chloro-3-methylcatechol, the major dioxygenation product formed from 3-chlorotoluene, is subject to quantitative dehalogenation after successive transformation by chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase, resulting in the formation of 2-methyldienelactone. 3-Chloro-5-methylcatechol is transformed to 2-chloro-4-methylmuconolactone.     

Isolation of an Asm− mutant of Rhizobium japonicum defective in symbiotic N2-fixation

Abstract A mutant strain of Rhizobium japonicum (CJ9) unable to assimilate ammonium (Asm⁻) was isolated following mutagenesis with N -methyl N -nitro-nitrosoguanidine (NTG). Glutamate synthase activity was not detectable in cell-free extracts of the mutant strain in contrast to the wild type and revertant strains. Although mutant CJ9 induced nitrogenase activity in an 'in vitro' assay system under microaerobic conditions, it failed to fix nitrogen (acetylene reduction) in soybean root nodules. These properties of mutant CJ9 constitute a new Asm⁻ mutant class in Rhizobium spp.     

The Modified β-Ketoadipate Pathway in Rhodococcus rhodochrous N75: Enzymology of 3-Methylmuconolactone Metabolism

Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified β-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native M_r of 128,000, and a subunit M_r of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.     

Role of nitrogen fixation in the autecology of Polaromonas naphthalenivorans in contaminated sediments

Polaromonas naphthalenivorans strain CJ2 is a Gram‐negative betaproteobacterium that was identified, using stable isotope probing in 2003, as a dominant in situ degrader of naphthalene in coal tar‐contaminated sediments. The sequenced genome of strain CJ2 revealed several genes conferring nitrogen fixation within a 65.6 kb region of strain CJ2's chromosome that is absent in the genome of its closest sequenced relative Polaromonas sp. strain JS666. Laboratory growth and nitrogenase assays verified that these genes are functional, providing an alternative source of nitrogen in N‐free media when using naphthalene or pyruvate as carbon sources. Knowing this, we investigated if nitrogen‐fixation activity could be detected in microcosms containing sediments from the field site where strain CJ2 was isolated. Inducing nitrogen limitation with the addition of glucose or naphthalene stimulated nitrogenase activity in amended sediments, as detected using the acetylene reduction assay. With the use of fluorescence microscopy, we screened the microcosm sediments for the presence of active strain CJ2 cells using a dual‐labelling approach. When we examined the carbon‐amended microcosm sediments stained with both a strain CJ2‐specific fluorescent in situ hybridization probe and a polyclonal fluorescently tagged antibody, we were able to detect dual‐labelled active cells. In contrast, in sediments that received no carbon addition (showing no nitrogenase activity), no dual‐labelled cells were detected. Furthermore, the naphthalene amendment enhanced the proportion of active strain CJ2 cells in the sediment relative to a glucose amendment. Field experiments performed in sediments where strain CJ2 was isolated showed nitrogenase activity in response to dosing with naphthalene. Dual‐label fluorescence staining of these sediments showed a fivefold increase in active strain CJ2 in the sediments dosed with naphthalene over those dosed with deionized water. These experiments show that nitrogen fixation may play an important role in naphthalene biodegradation by strain CJ2 and contribute to its ecological success.     

Effect of temperature, sodium chloride, and pH on growth of Listeria monocytogenes in cabbage juice.

Human illness and death have resulted from the consumption of milk, cheese, and cole slaw contaminated with Listeria monocytogenes. Since the effects of temperature, NaCl, and pH on the growth of the organism in cabbage were unknown, a series of experiments was designed to investigate these factors. Two strains (LCDC 81-861 and Scott A, both serotype 4b) were examined. At 30 degrees C, the viable population of the LCDC 81-861 strain increased in sterile unclarified cabbage juice (CJ) containing 0 to 1.5% NaCl; a decrease in the population of both strains occurred in juice containing greater than or equal to 2% NaCl. At 5 degrees C, the population of the Scott A strain in CJ containing up to 5% NaCl was reduced by about 90% over a 70-day period; the LCDC 81-861 strain was more sensitive to refrigeration but remained viable in CJ containing less than or equal to 3.5% NaCl for 70 days. Growth in CJ at 30 degrees C resulted in a decrease in pH from 5.6 to 4.1 within 8 days. Death of L. monocytogenes occurred at 30 degrees C when the organism was inoculated into sterile CJ adjusted to pH less than or equal to 4.6 with lactic acid. No viable cells were detected after 3 days at pH less than or equal to 4.2. At 5 degrees C, the rate of death at pH less than or equal to 4.8 was slower than at 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of temperature, sodium chloride, and pH on growth of Listeria monocytogenes in cabbage juice

Human illness and death have resulted from the consumption of milk, cheese, and cole slaw contaminated with Listeria monocytogenes. Since the effects of temperature, NaCl, and pH on the growth of the organism in cabbage were unknown, a series of experiments was designed to investigate these factors. Two strains (LCDC 81-861 and Scott A, both serotype 4b) were examined. At 30 degrees C, the viable population of the LCDC 81-861 strain increased in sterile unclarified cabbage juice (CJ) containing 0 to 1.5% NaCl; a decrease in the population of both strains occurred in juice containing greater than or equal to 2% NaCl. At 5 degrees C, the population of the Scott A strain in CJ containing up to 5% NaCl was reduced by about 90% over a 70-day period; the LCDC 81-861 strain was more sensitive to refrigeration but remained viable in CJ containing less than or equal to 3.5% NaCl for 70 days. Growth in CJ at 30 degrees C resulted in a decrease in pH from 5.6 to 4.1 within 8 days. Death of L. monocytogenes occurred at 30 degrees C when the organism was inoculated into sterile CJ adjusted to pH less than or equal to 4.6 with lactic acid. No viable cells were detected after 3 days at pH less than or equal to 4.2. At 5 degrees C, the rate of death at pH less than or equal to 4.8 was slower than at 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitrogen Fixation and Carbon Dioxide Assimilation in Rhizobium japonicum

In free-living Rhizobium japonicum cultures, the stimulatory effect of CO₂ on nitrogenase (acetylene reduction) activity was mediated through ribulose bisphosphate carboxylase activity. Two mutant strains (CJ5 and CJ6) of R. japonicum defective in CO₂ fixation were isolated by mitomycin C treatment. No ribulose bisphosphate carboxylase activity could be detected in strain CJ6, but a low level of enzyme activity was present in strain CJ5. Mutant strain CJ5 also exhibited pleiotropic effects on carbon metabolism. The mutant strains possessed reduced levels of hydrogen uptake, formate dehydrogenase, and phosphoribulokinase activities, which indicated a regulatory relationship between these enzymes. The CO₂-dependent stimulation of nitrogenase activity was not observed in the mutant strains. Both mutant strains nodulated soybean plants and fixed nitrogen at rates comparable to that of the wild-type strain.     

Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum

The glutamate dehydrogenase (gdh) gene of Escherichia coli was transferred into an ammonium assimilation deficient mutant (Asm^-) of Rhizobium japonicum (CJ9) using plasmid pRP301, a broad host range derivative of RP₄. Exconjugants capable of growth on ammonia as sole N-source occurred at a frequency of 6.8×10^-6. Assimilatory GDH (NADP⁺) activity was detected in the strain carrying the E. coli gdh gene and the pattern of ammonia assimilation via GDH was similar to that of the Asm⁺ wild type strain. However, GDH mediated ammonia assimilation was not subject to regulation by l-glutamate. Nitrogenase activity was expressed ex planta in R. japonicum CJ9 harbouring the gdh gene, however, the presence of the gdh gene did not restore symbiotic effectiveness to the CJ9 Asm^- strain in nodules. The gdh plasmid was maintained in approximately 90% of the isolates recovered from soybean nodules.Abbreviations gdh glutamate dehydrogenase - Asm^- mutant ammonia assimilation deficient mutant     

Identification of cis-diols as intermediates in the oxidation of aromatic acids by a strain of Pseudomonas putida that contains a TOL plasmid.

Pseudomonas putida BG1 was isolated from soil by enrichment with p-toluate and selection for growth with p-xylene. Other hydrocarbons that served as growth substrates were toluene, m-xylene, 3-ethyltoluene, and 1,2,4-trimethylbenzene. The enzymes responsible for growth on these substrates are encoded by a large plasmid with properties similar to those of TOL plasmids isolated from other strains of Pseudomonas. Treatment of P. putida BG1 with nitrosoguanidine led to the isolation of a mutant strain which, when grown with fructose, oxidized both p-xylene and p-toluate to (-)-cis-1,2-dihydroxy-4-methylcyclohexa-3,5-diene-1-carboxylic acid (cis-p-toluate diol). The structure of the diol was determined by conventional chemical techniques including identification of the products formed by acid-catalyzed dehydration and characterization of a methyl ester derivative. The cis-relative stereochemistry of the hydroxyl groups was determined by the isolation and characterization of an isopropylidene derivative. p-Xylene-grown cells contained an inducible NAD+-dependent dehydrogenase which formed catechols from cis-p-toluate diol and the analogous acid diols formed from the other hydrocarbon substrates listed above. The catechols were converted to meta ring fission products by an inducible catechol-2,3-dioxygenase which was partially purified from p-xylene-grown cells of P. putida BG1.     

β-Methylmuconolactone, a key intermediate in the dissimilation of methylaromatic compounds by a modified 3-oxoadipate pathway evolved in nocardioform actinomycetes

Abstract p -Toluate-grown cells of Rhodococcus ruber N75, R. corallinus N657, R. rhodochrous N5 and Rhodococcus strains BCN1, BCN2 and 4PH1 metabolized 4-methylcatechol by a modified 3-oxoadipate pathway. Steps in the conversion of this compound to 4-methyl-3-oxoadipic acid were investigated. The conversion of 4-carboxymethyl-3-methylbut-2-en-1, 4-olide to 4-carboxymethyl-3-methylbut-2-en-1, 4-olide by a new enzyme is described.     

Isolation and identification of two novel butenolides as products of dimethylbenzoate metabolism by Rhodococcus rhodochrous N75

Abstract 3,4-Dimethylbenzoic acid and 3,5-dimethylbenzoic acid were both oxidised by 4-methylbenzoate ( p -toluate)-grown cells of Rhodococcus rhodochrous N75 via the ortho -pathway through the intermediates 3,4- and 3,5-dimethylcatechol, respectively. Owing to the formation of the two novel dead-end metabolites, 4-carboxymethyl-2,3-dimethylbut-2-en-1,4-olide and 4-carboxymethyl-2,4-dimethylbut-2-en-1,4-olide from these substrates, 3,4- and 3,5-dimethylbenzoate did not serve as growth substrates for the strain.     

Construction of chimeric catechol 2,3-dioxygenase exhibiting improved activity against the suicide inhibitor 4-methylcatechol

Catechol 2,3-dioxygenase (C23O; EC 1.3.11.2), exemplified by XylE and NahH, catalyzes the ring cleavage of catechol and some substituted catechols. C23O is inactivated at an appreciable rate during the ring cleavage of 4-methylcatechol due to the oxidation of the Fe(II) cofactor to Fe(III). In this study, a C23O exhibiting improved activity against 4-methylcatechol was isolated. To isolate this C23O, diverse C23O gene sequences were PCR amplified from DNA which had been isolated from mixed cultures of phenol-degrading bacteria and subcloned in the middle of a known C23O gene sequence (xylE or nahH) to construct a library of chimeric C23O genes. These chimeric C23O genes were then introduced into Pseudomonas putida possessing some of the toluene catabolic genes (xylXYZLGFJQKJI). When a C23O gene (e.g., xylE) is introduced into this strain, the transformants cannot generally grow on p-toluate because 4-methylcatechol, a metabolite of p-toluate, is a substrate as well as a suicide inhibitor of C23O. However, a transformant of this strain capable of growing on p-toluate was isolated, and a chimeric C23O (named NY8) in this transformant was characterized. The rate of enzyme inactivation by 4-methylcatechol was lower in NY8 than in XylE. Furthermore, the rate of the reactivation of inactive C23O in a solution containing Fe(II) and ascorbic acid was higher in NY8 than in XylE. These properties of NY8 might allow the efficient metabolism of 4-methylcatechol and thus allow host cells to grow on p-toluate.     

Degradation of 2-methylaniline and chlorinated isomers of 2-methylaniline by Rhodococcus rhodochrous strain CTM

Rhodococcus rhodochrous strain CTM co-metabolized 2-methylaniline and some of its chlorinated isomers in the presence of ethanol as additional carbon source. Degradation of 2-methylaniline proceeded via 3-methylcatechol, which was metabolized mainly by meta-cleavage. In the case of 3-chloro-2-methylaniline, however, only a small proportion (about 10%) was subjected to meta-cleavage; the chlorinated meta-cleavage product was accumulated in the culture fluid as a dead-end metabolite. In contrast, 4-chloro-2-methylaniline was degraded via ortho-cleavage exclusively. Enzyme assays showed the presence of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase as inducible enzymes in strain CTM. Extended cultivation of strain CTM with 2-methylaniline and 3-chloro-2-methylaniline yielded mutants, including R. rhodochrous strain CTM2, that had lost catechol 2,3-dioxygenase activity; these mutants degraded the aromatic amines exclusively via the ortho-cleavage pathway. DNA hybridization experiments using a gene probe revealed the loss of the catechol 2,3-dioxygenase gene from strain CTM2.     

Dependence of transformation of chlorophenols by Rhodococci on position and number of chlorine atoms in the aromatic ring

Study of the conversion of chlorophenols by Rhodococcus opacus 1G, R. rhodnii 135, R. rhodochrous 89, and R. opacus 1cp disclosed the dependence of the conversion rate and pathway on the number and position of chlorine atoms in the aromatic ring. The most active chlorophenol converter, strain R. opacus 1cp, grew on each of the three isomeric monochlorophenols and on 2,4-dichlorophenol; the rate of growth decreased from 4-chlorophenol to 3-chlorophenol and then to 2-chlorophenol. The parameters of growth on 2,4-dichlorophenol were the same as on 3-chlorophenol. None of the strains studied utilized trichlorophenols. A detailed study of the pathway of chlorophenol transformation showed that 3-chloro-, 4-chloro-, and 2,4-dichlorophenol were utilized by the strains via a modified ortho-pathway. 2-Chlorophenol and 2,3-dichlorophenol were transformed by strains R. opacus 1cp and R. rhodochrous 89 via corresponding 3-chloro- and 3,4-dichloropyrocatechols, which were then hydroxylated with the formation of 4-chloropyrogallol and 4,5-dichloropyrogallol; this route had not previously been described in bacteria. Phenol hydroxylase of R. opacus 1G exhibited a previously undescribed catalytic pattern, catalyzing oxidative dehalogenation of 2,3,5-trichlorophenol with the formation of 3,5-dichloropyrocatechol but not hydroxylation of the nonsubstituted position 6.     

A gene cluster involved in degradation of substituted salicylates via ortho cleavage in Pseudomonas sp. strain MT1 encodes enzymes specifically adapted for transformation of 4-methylcatechol and 3-methylmuconate

Pseudomonas sp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.     

Cyclic (1 → 2)-β-d-glucans excreted by the glucuronan-producing strain Rhizobium meffloti M5N1CS CS (NCIM B 40472) and by the succinoglycan-producing strain Rhizobium meliloti M5N1

During fermentation, the mutant strain Rhizobium mefliloti M5N1 CS, which induces nodule formation on alfalfa roots, produces a partially acetylated (1 → 4)-β-d-glucuronan. In addition to this exopolysaccharide of high molecular weight, the mutant strain produces oligoglucoronates and cyclic (1 → 2)-β-d-glucans with degrees of polymerization from 17 to 30. Under the conditions applied, magnesium has no effect on cyclic glucan production by the mutant strain, but the succinoglycan production by the wild-type strain Rhizobium meliloti M5N1 increases.     

Transfer from Rhizobium japonicum to Azotobacter vinelandii of genes required for nodulation

A mutant strain of Azotobacter vinelandii that is unable to fix N2 (Nif-) was transformed to Nif+ with DNA from Rhizobium japonicum. Of 50 Nif+ transformants tested, 3 contained the O antigen-related polysaccharide that is present on the cell surface of a nodulating R. japonicum strain, but is absent from a non-nodulating mutant strain.