Evidence for a new pathway in the bacterial degradation of 4-fluorobenzoate

Six bacterial strains able to use 4-fluorobenzoic acid as their sole source of carbon and energy were isolated by selective enrichment from various water and soil samples from the Stuttgart area. According to their responses in biochemical and morphological tests, the organisms were assigned to the genera Alcaligenes, Pseudomonas, and Aureobacterium. To elucidate the degradation pathway of 4-fluorobenzoate, metabolic intermediates were identified. Five gram-negative isolates degraded this substrate via 4-fluorocatechol, as described in previous studies. In growth experiments, these strains excreted 50 to 90% of the fluoride from fluorobenzoate. Alcaligenes sp. strains RHO21 and RHO22 used all three isomers of monofluorobenzoate. Alcaligenes sp. strain RHO22 also grew on 4-chlorobenzoate. Aureobacterium sp. strain RHO25 transiently excreted 4-hydroxybenzoate into the culture medium during growth on 4-fluorobenzoate, and stoichiometric amounts of fluoride were released. In cell extracts from this strain, the enzymes for the conversion of 4-fluorobenzoate, 4-hydroxybenzoate, and 3,4-dihydroxybenzoate could be detected. All these enzymes were inducible by 4-fluorobenzoate. These data suggest a new pathway for the degradation of 4-fluorobenzoate by Aureobacterium sp. strain RHO25 via 4-hydroxybenzoate and 3,4-dihydroxybenzoate.     

Evolution of a new bacterial pathway for 4-nitrotoluene degradation

Bacteria that assimilate synthetic nitroarene compounds represent unique evolutionary models, as their metabolic pathways are in the process of adaptation and optimization for the consumption of these toxic chemicals. We used Acidovorax sp. strain JS42, which is capable of growth on nitrobenzene and 2-nitrotoluene, in experiments to examine how a nitroarene degradation pathway evolves when its host strain is challenged with direct selective pressure to assimilate non-native substrates. Although the same enzyme that initiates the degradation of nitrobenzene and 2-nitrotoluene also oxidizes 4-nitrotoluene to 4-methylcatechol, which is a growth substrate for JS42, the strain is incapable of growth on 4-nitrotoluene. Using long-term laboratory evolution experiments, we obtained JS42 mutants that gained the ability to grow on 4-nitrotoluene via a new degradation pathway. The underlying basis for this new activity resulted from the accumulation of specific mutations in the gene encoding the dioxygenase that catalyses the initial oxidation of nitroarene substrates, but at positions distal to the active site and previously unknown to affect activity in this or related enzymes. We constructed additional mutant dioxygenases to identify the order of mutations that led to the improved enzymes. Biochemical analyses revealed a defined, step-wise pathway for the evolution of the improved dioxygenases.     

Enzymatic formation, stability, and spontaneous reactions of 4-fluoromuconolactone, a metabolite of the bacterial degradation of 4-fluorobenzoate.

Enzymatic conversion of 4-fluorocatechol in the simultaneous presence of partially purified preparations of catechol 1,2-dioxygenase from Pseudomonas cepacia and muconate cycloisomerase from Alcaligenes eutrophus 335 yielded a product that was unambiguously identified as (+)-4-fluoromuconolactone [(+)-4-carboxymethyl-4-fluoro-but-2-en-4-olide]. This compound was shown to be the only major product formed from 3-fluoro-cis,cis-muconate by the action of muconate cycloisomerases from A. eutrophus 335, A. eutrophus JMP134, and P. cepacia as well as by the action of dichloromuconate cycloisomerase from A. eutrophus JMP134. This finding implies that dichloromuconate cycloisomerase, like the muconate cycloisomerases, catalyzes primarily a cycloisomerization reaction, which only in the case of chloro- and bromo-substituted substrates is connected to a dehalogenation. 4-Fluoromuconolactone at pH 7 decomposes by spontaneous reactions mainly to maleylacetate, which then decarboxylates to give cis-acetylacrylate. Although significant amounts of an unidentified compound are also formed from the fluorolactone, HF elimination to the two isomeric dienelactones (4-carboxymethylenebut-2-en-4-olides) is negligible. However, all spontaneous reactions proceed so slowly that an enzymatic conversion of 4-fluoromuconolactone must be assumed. Participation of dienelactone hydrolases in this reaction is indicated by their induction during growth of various strains with 4-fluorobenzoate. However, experiments with cell extracts of P. putida A3.12 suggest that at least one other hydrolytic enzyme is able to contribute to 4-fluoromuconolactone conversion. In light of these observations, earlier proposals for a 4-fluorobenzoate degradative pathway are discussed.     

Evidence for the aerobic degradation of tetrachloroethylene by a bacterial isolate

Summary A microorganism, which utilizes tetrachloroethylene (PCE) as a sole source of metabolic carbon under aerobic conditions, has been isolated. It has been identified as aPseudomonas spp. Degradation of PCE by this bacterium was quantified by gas chromatography.     

Evidence for a novel pathway in the degradation of fluorene by Pseudomonas sp. strain F274.

A fluorene-utilizing microorganism, identified as a species of Pseudomonas, was isolated from soil severely contaminated from creosote use and was shown to accumulate six major metabolites from fluorene in washed-cell incubations. Five of these products were identified as 9-fluorenol, 9-fluorenone, (+)-1,1a-dihydroxy-1-hydro-9-fluorenone, 8-hydroxy-3,4-benzocoumarin, and phthalic acid. This last compound was also identified in growing cultures supported by fluorene. Fluorene assimilation into cell biomass was estimated to be approximately 50%. The structures of accumulated products indicate that a previously undescribed pathway of fluorene catabolism is employed by Pseudomonas sp. strain F274. This pathway involves oxygenation of fluorene at C-9 to give 9-fluorenol, which is then dehydrogenated to the corresponding ketone, 9-fluorenone. Dioxygenase attack on 9-fluorenone adjacent to the carbonyl group gives an angular diol, 1,1a-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4-benzocoumarin and phthalic acid suggests that the five-membered ring of the angular diol is opened first and that the resulting 2'-carboxy derivative of 2,3-dihydroxy-biphenyl is catabolized by reactions analogous to those of biphenyl degradation, leading to the formation of phthalic acid. Cell extracts of fluorene-grown cells possessed high levels of an enzyme characteristic of phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase, together with protocatechuate 4,5-dioxygenase. On the basis of these findings, a pathway of fluorene degradation is proposed to account for its conversion to intermediary metabolites. A range of compounds with structures similar to that of fluorene was acted on by fluorene-grown cells to give products consistent with the initial reactions proposed.     

Anaerobic degradation of 2-fluorobenzoate by benzoate-degrading, denitrifying bacteria.

Three strains of anaerobically benzoate-degrading, denitrifying bacteria of the genus Pseudomonas were able to grow on 2-fluorobenzoate as the sole carbon and energy source. Fluoride ion release was stoichiometric, and the reduction of dissolved organic carbon indicated total degradation. Cells grown anaerobically with benzoate were adapted for immediate growth with 2-fluorobenzoate, and both compounds were substrates for an inducible benzoyl-coenzyme A synthetase, the initial enzyme of anaerobic degradation. It is proposed that fluoride is eliminated gratuitously by a regioselective reaction in a sequence common to both carbon sources. Benzoate, but not 2-fluorobenzoate, was oxidized by aerobically grown cells.     

Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2, 4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2, 6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2, 4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.     

New bacterial pathway for 4- and 5-chlorosalicylate degradation via 4-chlorocatechol and maleylacetate in Pseudomonas sp. strain MT1

Pseudomonas sp. strain MT1 is capable of degrading 4- and 5-chlorosalicylates via 4-chlorocatechol, 3-chloromuconate, and maleylacetate by a novel pathway. 3-Chloromuconate is transformed by muconate cycloisomerase of MT1 into protoanemonin, a dominant reaction product, as previously shown for other muconate cycloisomerases. However, kinetic data indicate that the muconate cycloisomerase of MT1 is specialized for 3-chloromuconate conversion and is not able to form cis-dienelactone. Protoanemonin is obviously a dead-end product of the pathway. A trans-dienelactone hydrolase (trans-DLH) was induced during growth on chlorosalicylates. Even though the purified enzyme did not act on either 3-chloromuconate or protoanemonin, the presence of muconate cylcoisomerase and trans-DLH together resulted in considerably lower protoanemonin concentrations but larger amounts of maleylacetate formed from 3-chloromuconate than the presence of muconate cycloisomerase alone resulted in. As trans-DLH also acts on 4-fluoromuconolactone, forming maleylacetate, we suggest that this enzyme acts on 4-chloromuconolactone as an intermediate in the muconate cycloisomerase-catalyzed transformation of 3-chloromuconate, thus preventing protoanemonin formation and favoring maleylacetate formation. The maleylacetate formed in this way is reduced by maleylacetate reductase. Chlorosalicylate degradation in MT1 thus occurs by a new pathway consisting of a patchwork of reactions catalyzed by enzymes from the 3-oxoadipate pathway (catechol 1,2-dioxygenase, muconate cycloisomerase) and the chlorocatechol pathway (maleylacetate reductase) and a trans-DLH.     

Evidence for a new, oxygen-regulated biosynthetic pathway for the pyrimidine moiety of thiamine in Salmonella typhimurium.

The synthesis of the pyrimidine moiety of thiamine (vitamin B1) shares five reactions with the de novo purine biosynthetic pathway. Aminoimidazole ribotide (AIR) is the last common intermediate before the two pathways diverge. Evidence for the existence of a new pathway to the pyrimidine which bypasses the de novo purine biosynthetic pathway is reported here. This pathway is only expressed under anaerobic growth conditions and is denoted alternative pyrimidine biosynthesis or APB. Labeling studies are consistent with pantothenate being a precursor to the pyrimidine moiety of thiamine that is synthesized by the APB pathway. The APB pathway is independent of the alternative purF function which was proposed previously (D. M. Downs and J. R. Roth, J. Bacteriol. 173:6597-6604, 1991). The alternative purF function is shown here to be affected by temperature and exogenous pantothenate. Although the evidence suggests that the APB pathway is separate from the alternative purF function, the relationship between this function and the APB pathway is not yet clear.     

Escherichia hermanii--a new bacterial strain for chlorobenzene degradation

A new bacterial strain capable of chlorobenzene degradation has been isolated from sludge of an industrial wastewater treatment plant. The micro-organism is short, rod-shaped, Gram-negative, yellow-pigmented and has been identified as Escherichia hermanii. It was observed that high chlorobenzene concentrations (up to 394 mg l-1) had low toxic effects towards this strain, which was able to degrade chlorobenzene without any previous adaptation.     

Acetone formation in the Vibrio family: a new pathway for bacterial leucine catabolism

There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of L-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. L-Leucine, but not D-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of L-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only alpha-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d(7))-L-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads.     

Anaerobic degradation of 4-methylbenzoate via a specific 4-methylbenzoyl-CoA pathway

The pathway for anaerobic degradation of 4-methylbenzoate was studied in the denitrifying alphaproteobacterium Magnetospirillum sp. strain pMbN1. Adaptation studies with whole cells indicated substrate-dependent induction of the capacity to degrade 4-methylbenzoate. Differential protein profiling (2D-DIGE) of 4-methylbenzoate- in comparison with benzoate- or succinate-adapted cells revealed the specific abundance increase of substrate-specific protein sets. Their coding genes form distinct clusters on the genome, two of which were assigned to 4-methylbenzoate and one to benzoate degradation. The predicted functions of the gene products agree with a specific 4-methylbenzoyl-CoA degradation pathway in addition to and analogous to the known anaerobic benzoyl-CoA degradation pathway. In vitro benzoyl-CoA and 4-methylbenzoyl-CoA reductase activities revealed the electron donor and ATP-dependent formation of the corresponding conjugated cyclic dienoyl-CoA/4-methyl-dienoyl-CoA products. The 4-methylbenzoyl-CoA reductase activity was induced in the presence of 4-methylbenzoate. In accordance, metabolite analysis of cultures grown with 4-methylbenzoate tentatively identified 4-methylcyclohex-1,5-diene-1-carboxylate. The 4-methylbenzoate induced genes were assigned to code for the putative 4-methylbenzoyl-CoA reductase; their products display pronounced sequence disparity from the conventional class I benzoyl-CoA reductase, which does not accept substituents at the para-position. Identification of 3-methylglutarate together with the formation of specific proteins for ring cleavage and β-oxidation in 4-methylbenzoate-adapted cells suggest conservation of the methyl group along the specific 4-methylbenzoyl-CoA degradation pathway.     

Effect of microtubular or translational inhibitors on general cell protein degradation. Evidence for a dual catabolic pathway.

Rat embryo fibroblasts were grown in Eagle's minimal essential medium with 10% serum and cell proteins prelabelled with L-[1-(14)C]leucine, followed by a 24h chase. When transferred to medium deprived of serum these cells showed a 2--3-fold increase in the production of trichloroacetic acid-soluble radioactivity during a 4h observation period. The microtubular poisons vinblastine, vincristine and colchicine partially inhibited this induced proteolysis, but had no effect on the proteolytic rate of cells maintained in medium with 10% serum. A similar discriminating effect on induced proteolysis was observed with cycloheximide, puromycin and insulin. The inhibitory effects of cycloheximide and vinblastine were not additive. These data support the hypothesis that, in addition to the basal turnover of cell proteins, a second mechanism of protein degradation involving cytoplasmic autophagy can be activated by nutritional step-down and is selectively inhibited by agents that interfere with microtubular function and protein synthesis.     

Evidence for a C4 NADP-ME photosynthetic pathway in Vetiveria zizanioides Stapf

ABSTRACT

Leaf anatomy (light and transmission electron microscopy), immunogold localization of Rubisco, photosynthetic enzyme activities, CO₂ assimilation and stomatal conductance were studied in Vetiveria zizanioides Stapf., a graminaceous plant native to tropical and subtropical areas, and cultivated in temperate climates (Northwestern Italy). Leaves possess a NADP-ME Kranz anatomy with bundle sheath cells containing chloroplasts located in a centrifugal position. Dimorphic chloroplasts were also observed; they are agranal and starchy in the bundle sheath and granal starchless in the mesophyll cells. Rubisco immunolocalization studies indicate that this enzyme occurs solely in the bundle sheath chloroplasts. Pyruvate-orthophosphate dikinase, NADP-dependent malate dehydrogenase (NADP-MDH), NADP-dependent malic enzyme (NADP-ME), PEP-carboxykinase and NAD-dependent malic enzyme (NAD-ME) activities were determined. Enzyme activity and some kinetic properties of NADP-ME and NADP-MDH as well as CO₂ compensation point and stomatal conductance values were calculated indicating a NADP-ME C₄ photosynthetic pathway. Biochemical and structural results indicate that V. zizanioides belongs to the C₄ NADP-ME variant. This plant appears to be well adapted to the varying environmental conditions typical of temperate climates, by retaining high enzyme activities and a low CO₂ compensation point.     

Intracellular degradation of the HIV-1 envelope glycoprotein. Evidence for, and some characteristics of, an endoplasmic reticulum degradation pathway.

Analysis of the fate of HIV-1 envelope protein gp160 (Env) has shown that newly synthesized proteins may be degraded within the biosynthetic pathway and that this degradation may take place in compartments other than the lysosomes. The fate of newly synthesized Env was studied in living BHK-21 cells with the recombinant vaccinia virus expression system. We found that gp160 not only undergoes physiological endoproteolytic cleavage, producing gp120, but is also degraded, producing proteolytic fragments of 120 kDa to 26 kDa in size, as determined by SDS/PAGE in non reducing conditions. Analysis of the 120-kDa proteolytic fragment, and comparison with gp120, showed that it is composed of peptides linked by disulfides bonds and lacks the V3-loop epitope and the C-terminal domain of gp120 (amino acids 506-516). A permeabilized cell system, with impaired transport of labeled Env from the endoplasmic reticulum (ER) to Golgi compartments, was developed to determine the site of degradation and to define some biochemical characteristics of the intracellular degradation process. In the semipermeable BHK-21 cells, there was: (a) no gp120 production (b), a progressive decrease in the amount of newly synthesized gp160 and a concomitant increase in the amount of a 120-kDa proteolytic fragment. This fragment had the same biochemical characteristics as the 120-kDa proteolytic fragment found in living nonpermeabilized cells, and (c) susceptibility of the V3 loop. This degradation process occurred in the ER, as shown by both biochemical and indirect immunofluorescence analysis. Furthermore, there was evidence that changes in redox state are involved in the ER-dependent envelope degradation pathway because adding reducing agents to permeabilized cells caused dose-dependent degradation of the 120-kDa proteolytic fragment and of the remaining gp160 glycoprotein. Thus our results provide direct evidence that regulated degradation of the HIV-1 envelope glycoprotein may take place in the ER of infected cells.     

Evidence for tyrosine-linked glycosaminoglycan in a bacterial surface protein.

The S-layer protein of Acetogenium kivui was subjected to proteolysis with different proteases and several high molecular mass glycosaminoglycan peptides containing glucose, galactosamine and an unidentified sugar-related component were separated by molecular sieve chromatography and reversed-phase HPLC and subjected to N-terminal sequence analysis. By methylation analysis glucose was found to be uniformly 1,6-linked, whereas galactosamine was exclusively 1,4-linked. Hydrazinolysis and subsequent amino-acid analysis as well as two-dimensional NMR spectroscopy were used to demonstrate that in these peptides carbohydrate was covalently linked to tyrosine. As all of the four Tyr-glycosylation sites were found to be preceded by valine, a new recognition sequence for glycosylation is suggested.     

Evidence for a new biologic pathway of androstenedione synthesis from 11-deoxycortisol

17-Hydroxyprogesterone is a well-known precursor of androstenedione in adrenal biosynthesis. This study using sheep adrenal incubations demonstrates that 11-deoxycortisol, the precursor of cortisol synthesis, also can be a precursor of androstenedione. Indeed, our data show that androstenedione synthesis is negatively correlated to the synthesis of cortisol and cortisone. This fact allowed us to infer that this new pathway is closely related to the activity of the 11 beta-hydroxylase that is responsible for the synthesis of cortisol. Indeed, when the activity of this enzyme is impaired, 11-deoxycortisol follows the pathway that leads to androstenedione synthesis in the adrenals. This pathway could explain, at least in part, the marked increase of androstenedione observed in congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency.