Putrescine N-acetyltransferase in Onchocerca volvulus and Ascaris suum, an enzyme which is involved in polyamine degradation and release of N-acetylputrescine.

A novel type of N-acetyltransferase, clearly different from the nuclear and cytosolic polyamine N-acetyltransferases of mammals, was recently found in the intestinal nematode Ascaris suum. The occurrence of this putrescine N-acetylating enzyme has also been noted in the filarial parasite Onchocerca volvulus. The enzyme was partially purified from adults of O. volvulus and A. suum by chromatography on DEAE-cellulose and cadaverine-Sepharose. Substrate specificities of the filarial enzyme resemble those of the N-acetyltransferase from A. suum, with respect to its preference for putrescine and other diamines above polyamines and histones. Additionally, both nematode enzymes acetylated histamine, whereas dopamine and serotonin were not accepted as substrates. The activities of the N-acetyltransferase from O. volvulus and A. suum were potently inhibited by the drug berenil; the type of inhibition was competitive with respect to putrescine. The inhibition constants for berenil were determined as 4.2 and 1.2 microM for the enzymes of O. volvulus and A. suum, the Km values for putrescine were found to be 330 microM and 250 microM, respectively. Putrescine N-acetyltransferase is discussed as a regulatory step in the degradation of excessive polyamines via polyamine oxidase to putrescine. At this branching point, putrescine is retained in the cell for de novo synthesis of spermidine and spermine, catabolized via diamine oxidase or acetylated to a suitable transport product for excretion.

Polyamine metabolism in filarial worms.

The human and animal filarial parasites Onchocerca volvulus, Dirofilaria immitis, Brugia patei and Litomosoides carinii contained low levels of putrescine but much higher levels of spermidine and spermine as estimated by ion-pair high pressure liquid chromatography; N-acetylated polyamines were present only in minute amounts. Enzyme activities of ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), respectively, were not detectable. Experiments carried out with O. volvulus and D. immitis demonstrated the uptake and bioconversion of labeled polyamines. There is evidence for the existence of a complete reverse pathway generating putrescine from spermidine and spermine, respectively, in both worms. N-Acetylating enzyme activities were detected in 100,000 X g preparations of homogenates from D. immitis which were capable to acetylate putrescine, spermidine and spermine. Long term incubation of the worms in the presence of labeled polyamines resulted in the excretion of putrescine and N-acetylputrescine.

Polyamine metabolism in Ancylostoma ceylanicum and Nippostrongylus brasiliensis.

Spermidine was detected as the major polyamine of Ancylostoma ceylanicum as well as Nippostrongylus brasiliensis. Spermine was present in lower amounts whereas the level of putrescine was even less. S-Adenosylmethionine decarboxylase, a rate-limiting enzyme in the biosynthetic pathway of polyamines, was demonstrated at low levels in both parasites. Decarboxylation of lysine and arginine was absent or negligible and that of ornithine questionable, as the enzyme activity was not inhibited by alpha-difluoromethylornithine while RMI 71,645, an irreversible inhibitor of ornithine aminotransferase, strongly inhibited the liberation of CO2 from ornithine. High activity of ornithine aminotransferase was observed in both the parasites and may interfere with the assay for ornithine decarboxylase. Adults of A. ceylanicum were found to rapidly take up spermidine and spermine from incubation medium while uptake of putrescine was very low. These results indicate that hookworms depend on uptake and interconversion rather than de novo synthesis for their polyamine requirement.

Metabolism of 3-methyldiphenyl ether by Sphingomonas sp. SS31.

The bacterium Sphingomonas sp. SS31, which was obtained from the diphenyl ether-degrading strain Sphingomonas sp. SS3 by an adaptation process, utilized 3-methyldiphenyl ether for growth in addition to diphenyl ether. The initial enzymatic attack onto this compound proceeded by a regioselective, but non-specific dioxygenation at the carbon carrying the ether bridge and the adjacent carbon of the unsubstituted as well as the methyl-substituted aromatic nucleus. Upon spontaneous decomposition, the resulting unstable hemiacetal structure yielded 3-methylphenol and catechol, or phenol, 3-methylcatechol, and 4-methylcatechol, respectively. Phenol and 3-methylphenol were oxidized to the corresponding catechols which, after subsequent ortho-cleavage, were channeled into the oxoadipate pathway.

Degradation of benzene 1,3-disulfonate by a mixed bacterial culture.

A benzene 1,3-disulfonate degrading mixed bacterial culture was isolated from the River Elbe downstream of Hamburg. The mixed culture was composed of five different bacterial strains. None of these strains grew in axenic culture with benzene 1,3-disulfonate as sole source of carbon and energy. In the presence of 4-nitrocatechol, resting cells of the mixed culture converted benzene 1,3-disulfonate to catechol 4-sulfonate. Experiments with cell-free extracts demonstrated that catechol 4-sulfonate was further metabolized via 3-sulfomuconate and 4-carboxymethyl-4-sulfobut-2-en-4-olide.

Transformation of 3-chlorodibenzofuran by Pseudomonas sp. HH69.

The dibenzofuran-degrading bacterial strain Pseudomonas sp. HH69 showed high oxidative activity towards 3-chlorodibenzofuran (3CDF). During the co-metabolic turnover of 3CDF large amounts of 4-chlorosalicylate and temporarily small amounts of salicylate were excreted. Simultaneously a yellow colour appeared due to the excretion of two polar products. Conversion of 3CDF by a mutant, derived from Pseudomonas sp. HH69 and defective in 2,3-dihydroxybiphenyl-1,2-dioxygenase led to the formation of equal quantities of 4'-chloro-2,2',3-trihydroxybiphenyl (4'CTHBP) and 4-chloro-2,2',3-trihydroxybiphenyl (4CTHBP). Crude extracts of the wild type transformed 4'CTHBP to 4-chlorosalicylate, whilst 4CTHBP was transformed to salicylate. Hence, we propose a non-selective initial attack on both aromatic rings of 3CDF and a degradative pathway for the resulting chlorotrihydroxybiphenyls.

Interaction of Sphingomonas and Pseudomonas strains in the degradation of chlorinated dibenzofurans.

We have studied the concerted degradation of two monochlorodibenzofurans by a bacterial consortium, consisting of the chlorodibenzofurans-cometabolizing and chlorosalicylates-excreting strain Sphingomonas sp RW16, and Pseudomonas sp RW10, which mineralized the released chlorosalicylates. Neither of the organisms was able to grow with chlorodibenzofurans alone. Degradation of 2-chloro- and 3-chlorodibenzofuran proceeded to the end products 5-chloro- and 4-chlorosalicylate, respectively, when the initial dioxygenase of Sphingomonas sp RW 16 attacked the unchlorinated aromatic ring of the heterocyclic dibenzofuran molecule. 2-Hydroxypenta-2,4-dienoate, formed upon meta-cleavage of the intermediary chlorotrihydroxybiphenyls, served as a growth substrate for the sphingomonad. Presumably, most of the chlorosalicylates were excreted and degraded further by Pseudomonas sp RW10. Mineralization of both chlorosalicylates proceeded through a converging pathway, via 4-chlorocatechol, and protoanemonin. Chlorosalicylates were mineralized by the pseudomonad only when their concentration in the culture medium was below 1.5 mM. In the case of initial dioxygenation taking place on the chlorinated aromatic ring, salicylate and chlorinated hydroxypentadienoates should be formed. The metabolic fate of putative chlorohydroxypentadienoates is not clear; ie, they may be channeled into unproductive catabolism and, thus, represent the critical point in the breakdown of the carbon of these two chlorodibenzofurans by Sphingomonas sp RW16.

The polyamine metabolism of filarial worms as chemotherapeutic target.

Parasite-specific putrescine-N-acetyltransferase and polyamine oxidase, both involved in the reversed pathway of polyamine metabolism, were demonstrated for Ascaris suum and Onchocerca volvulus. Berenil-treatment was found to be correlated with accumulation of polyamines, especially spermine, obviously due to blockaded polyamine interconversion. Furthermore it was shown that added spermine to the culture medium led to the death of worms. These specificities might be exploited for chemotherapy of filarial infections. Polyamines are widely distributed in the nature. They are found in higher and lower eucaryotes and in procaryotes as well as in viruses (Tabor and Tabor, 1984). During the last years there have been many approaches to examine the role of polyamines in cell growth and differentiation in vertebrates (Tabor and Tabor, 1984; Pegg, 1986). The polyamine metabolism of parasites also has attracted increasing interest, e.g. in African trypanosomes the initial enzyme of polyamine synthesis - ornithine decarboxylase - has been exploited as a target for chemotherapy by using DFMO (DL alpha-difluoromethylornithine) (Bacchi et al., 1980; Bacchi et al., 1983; Fairlamb et al., 1985; Giffin et al., 1986). The polyamine metabolism of filaria and other helminths is still a neglected area of research, although there are reports about distribution pattern of polyamines and some peculiarities of polyamine metabolism in filarial worms (Srivastava et al., 1980; Wittich et al., 1987; Walter, 1988). DFMO and MGBG (methylglyoxal bis-(guanylhydrazone], both of which are potent inhibitors of polyamine synthesis in mammals, do not significantly effect the viability of filarial worms (Wittich et al., 1987).(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation of dioxin-like compounds by microorganisms.

Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF; PCDD/F, dioxins) have not been commercially produced in bulk amounts, as were polychlorinated biphenyls and other haloaromatic organics. Within the past two decades a lot of information has accumulated on the biodegradation of PCDD/F and other dioxin-like compounds because of their toxicity and because of significant environmental concern about many congeners of this class of chemicals. PCDD/F are subjected to reductive dehalogenations leading to less halogenated congeners, which can be attacked efficiently by fungal and bacterial oxidases and dioxygenases. In several cases these compounds can be utilized as carbon and energy sources. Pathways for their enzymatic degradation and the organisation of the corresponding degradative genes have been elucidated. Consequently, biotechnological applications will exploit the degradative potential of such microorganisms for bioremediation of contaminated sites.

A novel type of putrescine (diamine)-acetylating enzyme from the nematode Ascaris suum.

A cytosolic enzyme catalysing the acetylation of the diamines putrescine, cadaverine, 1,3-diaminopropane and 1,6-diaminohexane has been partially purified from reproductive tissue of the intestinal parasitic nematode Ascaris suum. The enzyme formed N-acetylated derivatives of the above diamines when incubated in the presence of acetyl-CoA. The Michaelis constants (Km) for the above diamines were 0.25 nM, 0.1 mM, 1.25 mM and 0.4 mM respectively, and the apparent Km for acetyl-CoA was 7.7 microM. sym-Norspermidine was also acetylated by this enzyme preparation, and, at a much lower rate, the enzyme acted on sym-norspermine. The common polyamines, spermidine and spermine, and histones were not substrates. Purification steps involved a freezing-and-thawing procedure to release enzyme activity from unknown inhibitors, DEAE-cellulose chromatography and affinity chromatography on cadaverine-Sepharose, from which the enzyme was eluted by increasing ionic strength. The enzyme exhibited an apparent Mr of about 38,000-40,000, and it consisted of at least two subunits, of which the catalytic one had an Mr of about 13,000. The partially purified enzyme showed no deacetylase activity, and its activity was competitively inhibited by the product N-acetylputrescine, but not by CoA. The name putrescine N-acetyltransferase is suggested for this enzyme, which may have an important function in the degradation of diamines of lower eukaryotes.

Degradation of naphthalene-2,6- and naphthalene-1,6-disulfonic acid by a Moraxella sp.

A naphthalene-2,6-disulfonic acid (2,6NDS)-degrading Moraxella strain was isolated from an industrial sewage plant. This culture could also be adapted to naphthalene-1,6-disulfonic acid as growth substrate. Regioselective 1,2-dioxygenation effected desulfonation and catabolism to 5-sulfosalicylic acid (5SS), which also could be used as the sole carbon source. 5SS-grown cells exhibited high gentisate 1,2-dioxygenase activity. Neither 5SS- nor gentisate-grown cells oxidized 2,6NDS; therefore, 2,6NDS or an early metabolite must serve as an inducer of the initial catabolic enzyme(s).

Chromosomal integration of tcb chlorocatechol degradation pathway genes as a means of expanding the growth substrate range of bacteria to include haloaromatics.

The tcbR-tcbCDEF gene cluster, coding for the chlorocatechol ortho-cleavage pathway in Pseudomonas sp. strain P51, has been cloned into a Tn5-based minitransposon. The minitransposon carrying the tcb gene cluster and a kanamycin resistance gene was transferred to Pseudomonas putida KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance. Transconjugants able to utilize 3-chlorobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants. The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chlorobenzoate. Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance possessed a single copy. Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb gene cluster was apparent. Introduction of two copies of the tcb gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source. Expression of the tcb chlorocatechol catabolic operon in P. putida thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates.

Filaricidal effect of mefloquine on adults and microfilariae of Brugia patei and Brugia malayi.

Mefloquine, DL-erythro-2,8-bis(trifluoromethyl)-alpha-(2-piperidyl)-4-quinoline methanolhydrochloride, a recently developed antimalarial drug shows filaricidal activity against larval and adult stages of Brugia patei and B. malayi maintained "in vitro". In the concentration range of 10 to 2 microM mefloquine paralysed and killed the filarial worms within 10 h and 3 d, respectively. The lethal effect of mefloquine treatment on larval and adult worms was shown by loss of motility as well as by decrease of lactate excretion by adults. Chloroquine at a concentration of 10 microM did not affect motility and survival of microfilariae and adults of B. patei. Addition of serum to the cultivation medium abolished the filaricidal effect, possibly due to the tight binding of mefloquine to serum proteins, thereby affecting the uptake of the drug into the parasite.

Chlorocatechols substituted at positions 4 and 5 are substrates of the broad-spectrum chlorocatechol 1,2-dioxygenase of Pseudomonas chlororaphis RW71.

The nucleotide sequence of a 10,528-bp region comprising the chlorocatechol pathway gene cluster tetRtetCDEF of the 1,2,3,4-tetrachlorobenzene via the tetrachlorocatechol-mineralizing bacterium Pseudomonas chlororaphis RW71 (T. Potrawfke, K. N. Timmis, and R.-M. Wittich, Appl. Environ. Microbiol. 64:3798-3806, 1998) was analyzed. The chlorocatechol 1,2-dioxygenase gene tetC was cloned and overexpressed in Escherichia coli. The recombinant gene product was purified, and the alpha,alpha-homodimeric TetC was characterized. Electron paramagnetic resonance measurements confirmed the presence of a high-spin-state Fe(III) atom per monomer in the holoprotein. The productive transformation by purified TetC of chlorocatechols bearing chlorine atoms in positions 4 and 5 provided strong evidence for a significantly broadened substrate spectrum of this dioxygenase compared with other chlorocatechol dioxygenases. The conversion of 4,5-dichloro- or tetrachlorocatechol, in the presence of catechol, displayed strong competitive inhibition of catechol turnover. 3-Chlorocatechol, however, was simultaneously transformed, with a rate similar to that of the 4,5-halogenated catechols, indicating similar specificity constants. These novel characteristics of TetC thus differ significantly from results obtained from hitherto analyzed catechol 1,2-dioxygenases and chlorocatechol 1,2-dioxygenases.

Biodegradation and transformation of 4,4'- and 2,4-dihalodiphenyl ethers by Sphingomonas sp. strain SS33.

The bacterium Sphingomonas sp. strain SS33, obtained from parent diphenyl ether-mineralizing strain SS3 (S. Schmidt, R.-M. Wittich, D. Erdmann, H. Wilkes, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:2744-2750, 1992) after several weeks of adaptation on 4,4'-difluorodiphenyl ether as the new target compound, also utilized 4,4'-dichlorodiphenyl ether for growth. Intermediary halocatechols were also mineralized via the ortho pathway by type I enzymes. 4,4'-Dibromodiphenyl ether was not used as a carbon source although transformation by resting cells yielded mononuclear haloaromatic compounds, such as 4-bromophenol and 4-bromocatechol. The same was true for the conversion of 2,4-dichlorodiphenyl ether, which yielded the respective (halo-) phenols and (halo-) catechols.

Ascaris suum and Onchocerca volvulus: S-adenosylmethionine decarboxylase.

Putrescine-dependent S-adenosylmethionine decarboxylase (EC 4.1.1.50) was demonstrated in Ascaris suum and Onchocerca volvulus; activation was found to be about fourfold by putrescine. Mg2+ did not affect the enzyme activity. A. suum was taken as a model nematode and its S-adenosylmethionine decarboxylase was partially purified and characterized. The molecular weight was estimated to be 220,000. The apparent Km-value for adenosylmethionine was determined to be 17 microM. Methylglyoxal bis(guanylhydrazone) and berenil competitively inhibited the enzyme activity; the apparent Ki-values were found to be 0.24 microM and 0.11 microM, respectively. The dependence of filarial worms on uptake and interconversion of putrescine and polyamines as well as properties of the S-adenosylmethionine decarboxylase, different from the host enzyme, points to the polyamine metabolisms as a useful target for chemotherapy.

A functional 4-hydroxysalicylate/hydroxyquinol degradative pathway gene cluster is linked to the initial dibenzo-p-dioxin pathway genes in Sphingomonas sp. strain RW1.

The bacterium Sphingomonas sp. strain RW1 is able to use dibenzo-p-dioxin, dibenzofuran, and several hydroxylated derivatives as sole sources of carbon and energy. We have determined and analyzed the nucleic acid sequence of a 9,997-bp HindIII fragment downstream of cistrons dxnA1A2, which encode the dioxygenase component of the initial dioxygenase system of the corresponding catabolic pathways. This fragment contains 10 colinear open reading frames (ORFs), apparently organized in one compact operon. The enzymatic activities of some proteins encoded by these genes were analyzed in the strain RW1 and, after hyperexpression, in Escherichia coli. The first three ORFs of the locus, designated dxnC, ORF2, and fdx3, specify a protein with a low homology to bacterial siderophore receptors, a polypeptide representing no significant homology to known proteins, and a putative ferredoxin, respectively. dxnD encodes a 69-kDa phenol monooxygenase-like protein with activity for the turnover of 4-hydroxysalicylate, and dxnE codes for a 37-kDa protein whose sequence and activity are similar to those of known maleylacetate reductases. The following gene, dxnF, encodes a 33-kDa intradiol dioxygenase which efficiently cleaves hydroxyquinol, yielding maleylacetate, the ketoform of 3-hydroxy-cis,cis-muconate. The heteromeric protein encoded by dxnGH is a 3-oxoadipate succinyl coenzyme A (succinyl-CoA) transferase, whereas dxnI specifies a protein exhibiting marked homology to acetyl-CoA acetyltransferases (thiolases). The last ORF of the sequenced fragment codes for a putative transposase. DxnD, DxnF, DxnE, DxnGH, and DxnI (the activities of most of them have also been detected in strain RW1) thus form a complete 4-hydroxysalicylate/hydroxyquinol degradative pathway. A route for the mineralization of the growth substrates 3-hydroxydibenzofuran and 2-hydroxydibenzo-p-dioxin in Sphingomonas sp. strain RW1 thus suggests itself.

Biodegradation of diphenyl ether and its monohalogenated derivatives by Sphingomonas sp. strain SS3.

The bacterium Sphingomonas sp. strain SS3, which utilizes diphenyl ether and its 4-fluoro, 4-chloro, and (to a considerably lesser extent) 4-bromo derivatives as sole sources of carbon and energy, was enriched from soil samples of an industrial waste deposit. The bacterium showed cometabolic activities toward all other isomeric monohalogenated diphenyl ethers. During diphenyl ether degradation in batch culture experiments, phenol and catechol were produced as intermediates which were then channeled into the 3-oxoadipate pathway. The initial step in the degradation follows the recently discovered mechanism of 1,2-dioxygenation, which yields unstable phenolic hemiacetals from diphenyl ether structures. Oxidation of the structure-related dibenzo-p-dioxin yielded 2-(2-hydroxyphenoxy)-muconate upon ortho cleavage of the intermediate 2,2',3-trihydroxydiphenyl ether. Formation of phenol, catechol, halophenol, and halocatechol from the conversion of monohalogenated diphenyl ethers gives evidence for a nonspecific attack of the dioxygenating enzyme system.

Degradation of 1,2,4-trichloro- and 1,2,4,5-tetrachlorobenzene by pseudomonas strains.

Two Pseudomonas sp. strains, capable of growth on chlorinated benzenes as the sole source of carbon and energy, were isolated by selective enrichment from soil samples of an industrial waste deposit. Strain PS12 grew on monochlorobenzene, all three isomeric dichlorobenzenes, and 1,2,4-trichlorobenzene (1,2,4-TCB). Strain PS14 additionally used 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB). During growth on these compounds both strains released stoichiometric amounts of chloride ions. The first steps of the catabolism of 1,2,4-TCB and 1,2,4,5-TeCB proceeded via dioxygenation of the aromatic nuclei and furnished 3,4,6-trichlorocatechol. The intermediary cis-3,4,6-trichloro-1,2-dihydroxycyclohexa-3,5-diene (TCB dihydrodiol) formed from 1,2,4-TCB was rearomatized by an NAD-dependent dihydrodiol dehydrogenase activity, while in the case of 1,2,4,5-TeCB oxidation the catechol was obviously produced by spontaneous elimination of hydrogen chloride from the initially formed 1,3,4,6-tetrachloro-1,2-dihydroxycyclohexa-3,5-diene. Subsequent ortho cleavage was catalyzed by a type II catechol 1,2-dioxygenase producing the corresponding 2,3,5-trichloromuconate which was channeled into the tricarboxylic acid pathway via an ordinary degradation sequence, which in the present case included 2-chloro-3-oxoadipate. From the structure-related compound 2,4,5-trichloronitrobenzene the nitro group was released as nitrite, leaving the above metabolite as 3,4,6-trichlorocatechol. Enzyme activities for the oxidation of chlorobenzenes and halogenated metabolites were induced by both strains during growth on these haloaromatics and, to a considerable extent, during growth of strain PS12 on acetate.

An extractive membrane biofilm reactor for degradation of 1,3-dichloropropene in industrial waste water.

A bacterial biofilm, capable of mineralising a technical mixture of cis- and trans-1,3-dichloropropene (DCPE), was enriched on the biomedium side of an extractive membrane biofilm reactor (EMBR). The membrane separates the biomedium from the industrial waste water, in terms of pH, ionic strength and the concentration of toxic chemicals. The biofilm, attached to a silicone membrane, is able to mineralize DCPE after its diffusion through the membrane. Five bacterial strains with degradation capabilities were isolated from the metabolically active biofilm and further investigated in batch experiments. Two of them, Rhodococcus erythropolis strains EK2 and EK5, can grow with DCPE as the sole carbon source. Pseudomonas sp. EK1 uzilizes cis-3-chloroallylalcohol and cis-3-chloroacrylic acid, whereas the metabolite trans-3-chloroacrylic acid represents a dead-end product of the pathway of this strain. The other two strains, Delftia sp. EK3 and EK4, although unable to grow with DCPE as the carbon source, can transform DCPE and its upper-pathway intermediates at reasonable conversion rates. They may represent helper functions of the biofilm consortium, which mineralised up to 12.5 mmol DCPE per hour per gram of biomass protein. Higher feed rates in the EMBR (up to 15 mmol per hour per 100-l bioreactor volume) and shock loads corresponding to concentrations up to 1.8 mmol l-1 led to a significant increase in the freely floating bacterial biomass in the reactor medium (OD546 = 0.2). At the standard operating feed rate of 1.8 mmol h-1, the free biomass concentration was very low (OD546 = 0.04).