Utilization of complex phenolic compounds by Acinetobacter sp.

Summary Acinetobacter sp. utilized the [ring-¹⁴C]dehydropolymer of coniferyl alcohol (DHP) (sp. act. 1.4 × 10⁴ dpm/mg), ¹⁴C-labelled teakwood lignin (sp. act. 2.5 × 10⁴ dpm/mg), guaiacolglyceryl ether, 2-methoxy-4-formylphenoxyacetic acid, p-benzyloxyphenol, dehydrodivanillyl alcohol, dehydrodiisoeugenol, veratrylglycerol--guaiacyl ether, conidendrin, black liquor lignin and indulin as sole carbon sources. The bacterium produced p-coumaric acid, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid and catechol as intermediates from lignins. Acinetobacter sp. produced catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase during the degradation of lignins. Correspondence to: A. Mahadevan     

Degradation of labelled lignins and veratrylglycerol-beta-guaiacyl ether by Acinetobacter sp

Acinetobacter sp. evolved 14CO2 from 14C-(ring)DHP lignin and 14C-teakwood lignin. Veratrylglycerol-beta-guaiacyl ether, a lignin model compound with beta-o-4 linkage was cleaved by Acinetobacter sp. Veratrylglycerol-beta-guaiacyl ether into 2(o-methoxyphenoxy) ethanol and veratrylalcohol 2(o-methoxyphenoxy) ethanol was degraded to guaiacol and then to catechol whereas veratrylalcohol was converted to veratraldehyde, veratric acid, vanillic acid, protocatechuic acid and catechol. Both catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase were detected in veratrylglycerol-beta-guaiacyl ether grown cultures.     

Degradation of p-benzyloxyphenol by Acinetobacter sp.

Abstract Acinetobacter sp. utilized p -benzyloxyphenol as sole carbon source and degraded it to p -hydroxybenzaldehyde, p -hydroxybenzoic acid, protocatechuic acid and catechol. The intermediates were identified by paper chromatography, TLC, IR, GC and HPLC. Acinetobacter sp. produced protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase during the degradation of p -benzoloxyphenol.     

Degradation of indulin by Candida albicans.

Candida albicans utilized 14C (ring) labelled dehydropolymer of coniferyl alcohol, 14C-teakwood lignin and indulin and released p-hydroxybenzoic acid, vanillic acid, 3,4-dihydroxybenzoic acid and catechol as by products from lignin. Candida albicans produced catechol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase, intra- and extracellular polyphenol oxidase and peroxidase during indulin degradation. The study suggests that Candida albicans degrades different types of lignin.     

Chemical intervention in bacterial lignin degradation pathways: Development of selective inhibitors for intradiol and extradiol catechol dioxygenases

Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC₅₀ 15 μM) and D3 for MhpB (IC₅₀ 110 μM). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1 mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the β-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde.     

Catabolism of Substituted Benzoic Acids by Streptomyces Species

Four thermotolerant actinomycetes from soil, identified as Streptomyces albulus 321, Streptomyces sioyaensis P5, Streptomyces viridosporus T7A, and Streptomyces sp. V7, were grown at 45°C in media containing either benzoic acid or hydroxyl- and methoxyl-substituted benzoic acids as the principal carbon sources. Benzoic acid was converted to catechol; p-hydroxybenzoic, vanillic, and veratric acids were converted to protocatechuic acid; and m-hydroxybenzoic acid was converted to gentisic acid. Catechol, protocatechuic acid, and gentisic acid were cleaved by catechol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase, and gentisate 1,2-dioxygenase, respectively. Dioxygenases appeared only in induced cultures. m-Hydroxybenzoic, m-anisic, and p-anisic acids were gratuitous inducers of dioxygenases in some strains. One strain converted vanillic acid to guaiacol.     

Degradation of aromatic compounds byRhizobium spp.

Summary The ability of rhizobia to utilize catechol, protocatechuic acid, salicylic acid, p-hydroxybenzoic acid and catechin was investigated. The degradation pathway of p-hydroxybenzoate byRhizobium japonicum, R. phaseoli, R. leguminosarum, R. trifolii andRhizobium sp. isolated from bean was also studied.R. leguminosarum, R. phaseoli andR. trifolii metabolized p-hydroxybenzoate to protocatechuate which was cleaved by protocatechuate 3,4-dioxygenasevia ortho pathway.R. japonicum degraded p-hydroxybenzoate to catechol which was cleaved by catechol 1,2-dioxygenase.Rhizobium sp., a bean isolate, dissimilatedp-hydroxybenzoate to salicylate. Salicylate was converted to gentisic acid prior to ring cleavage. The rhizobia convertedp-hydroxybenzoate to Rothera positive substance. Catechol and protocatechuic acid were directly cleaved by the species.R. japonicum converted catechin to protocatechuic acid.     

Degradation of coniferyl alcohol and other lignin-related aromatic compounds by Nocardia sp. DSM 1069

Nocardia sp. DSM 1069 was grown on mineral salt media with coniferyl alcohol, 4-methoxybenzoic acid, 3-methoxybenzoic acid or veratric acid as sole sources of carbon and energy. During incubation on coniferyl alcohol, the formation of coniferyl aldehyde, ferulic acid and quantitative accumulation of vanillic acid and proteocatechuic acid could be achieved with mutants. Washed cell suspensions of N. sp. grown on 4-methoxybenzoic acid, oxidized 4-hydroxybenzoic acid and protocatechuic acid. Cells grown on veratric acid, oxidized vanillic acid, isovanillic acid, and protocatechuic acid. Cell extracts were shown to cleave protocatechuic acid by ortho-fission.A mutant without protocatechuate 3,4-dioxygenase activity was not influenced in its growth on 3 methoxybenzoic acid. Cell free extracts of cells grown on 3-methoxybenzoic acid were shown to catalyze the oxidation of gentisic acid (2,5-dihydroxybenzoic acid). The resulting ring cleavage product was further metabolized by a glutathione dependent reaction.The specificity of the demethylation reactions has been investigated with a mutant unable to grow on vanillic acid. This mutant was not impaired in the degradation of isovanillic acid, 4-methoxy-, or 3-methoxybenzoic acid, whereas growth of this mutant on veratric acid (3,4-dimethoxybenzoic acid) was only half as much as that of the wild type. Concomitantly with growth on veratric acid this mutant accumulated vanillic acid with a yield of about 50%.A pathway for the catabolism of coniferyl alcohol, involving oxidation and shortening of the side chain, and of 4-methoxybenzoic acid and veratric acid with protocatechuic acid as intermediate is being proposed. A second one is proposed for the degradation of 3-methoxybenzoic acid with gentisic acid as intermediate.     

Metabolism of cinnamic, p-coumaric, and ferulic acids by Streptomyces setonii

Streptomyces setonii strain 75Vi2 was grown at 45 degrees C in liquid media containing yeast extract and trans-cinnamic acid, p-coumaric acid, ferulic acid, or vanillin. Gas chromatography, thin-layer chromatography, and mass spectrometry showed that cinnamic acid was catabolized via benzaldehyde, benzoic acid, and catechol; p-coumaric acid was catabolized via p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and protocatechuic acid; ferulic acid was catabolized via vanillin, vanillic acid, and protocatechuic acid. When vanillin was used as the initial growth substrate, it was catabolized via vanillic acid, guaiacol, and catechol. The inducible ring-cleavage dioxygenases catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase were detected with an oxygen electrode in cell-free extracts of cultures grown in media with aromatic growth substrates and yeast extract.     

倭蜂猴粪便微生物苯酚羟化酶和邻苯二酚1,2-双加氧酶基因多样性研究

【目的】分析倭蜂猴粪便微生物中苯酚羟化酶(Phenol hydroxylase,PH)和邻苯二酚1,2-双加氧酶(Catechol 1,2-dioxygenase,C12O)的基因多样性。【方法】利用简并引物,以倭蜂猴粪便微生物宏基因组DNA为模板,通过PCR扩增,分别构建PH和C12O基因克隆文库,并对克隆进行测序分析。【结果】倭蜂猴粪便微生物来源的PH和C12O基因序列经BLAST比对分析,与GenBank中相应酶的序列一致性分别介于92%?100%和87%?100%。系统进化树分析表明PH基因序列与Neisseria、Burkholderia、Alcaligenes、Acinetobacter 4个属来源的PH序列相关;C12O基因序列全部与Acinetobacter来源的C12O序列相关。序列比对结果表明PH序列具有LmPH (Largest subunit of multicomponent PH)中高保守的两个DEXRH结构域;C12O序列具有能被Ag+和Hg2+抑制的位点(半胱氨酸)。【结论】倭蜂猴粪便微生物来源的PH为多组分PH,其降解苯酚的中间产物邻苯二酚可以被C12O通过邻位开环途径裂解。     

Degradation of naphthalene by thermophilic bacteria <Emphasis Type="Italic">via</Emphasis> a pathway,through protocatechuic acid

A number of thermophilic bacteria capable of utilizing naphthalene as a sole source of carbon were isolated from a high-temperature oilfield in Lithuania. These isolates were able to utilize several other aromatic compounds, such as anthracene, benzene, phenol, benzene-1, 3-diol, protocatechuic acid as well. Thermophilic isolate G27 ascribed to Geobacillus genus was found to have a high aromatic compound degrading capacity. Spectrophotometric determination of enzyme activities in cell-free extracts revealed that the last aromatic ring fission enzyme in naphthalene biotransformation by Geobacillus sp. G27 was inducible via protocatechuate 3, 4-dioxygenase; no protocatechuate 4, 5-dioxygenase, protocatechuate 2, 3-dioxygenase activities were detected. Intermediates such as o-phthalic and protocatechuic acids detected in culture supernatant confirmed that the metabolism of naphthalene by Geobacillus sp. G27 can proceed through protocatechuic acid via ortho-cleavage pathway and thus differs from the pathways known for mesophilic bacteria.     

Peptide mass fingerprinting- and 2-DE/MS-based analysis of the biodegradation potential for monocyclic aromatic hydrocarbons in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

The combined analysis of peptide mass fingerprinting and 2-DE/MS using the induced and selected protein spots following growth of Pseudomonas sp. DU102 on benzoate or p-hydroxybenzoate revealed not only alpha- and beta-subunits of protocatechuate 3,4-dioxygenase but also catechol 1,2-dioxygenase responsible for ortho-pathway through ring-cleavage of aromatic compounds. Toluate 1,2-dioxygenase and p-hydroxybenzoate hydroxylase were also identified. Purification of intradiol dioxygenases such as catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase from the benzoate or p-hydroxybenzoate culture makes it possible to trace the biodegradation pathway of strain DU102 for monocyclic aromatic hydrocarbons. Interestingly, vanillin-induced protocatechuate 3,4-dioxygenase was identical in amino acid sequences with protocatechuate 3,4-dioxygenase from p-hydroxybenzoate.     

一株菲降解细菌的分离鉴定及其特性

通过选择性富集培养,从沈抚灌区石油污染土壤中分离到1株菲降解细菌.试验证明该菌株能以菲为唯一碳源和能源生长.经形态学、生理生化鉴定和16S rRNA基因序列比对分析,确定该菌株属于不动杆菌属,命名为Acinetobacter sp. L2. 系统发育进化分析发现,L2菌株与Acinetobacter sp. DG880［AY258108］亲源关系最近.L2菌株培养7 d后对菲的降解率达96.3%.邻苯二酚2,3-双加氧酶活力测定表明,L2菌株可能含有菲降解基因.     

草果果实中的酚性成分

从草果( Amomum tsao-ko) 果实的甲醇提取物中分离得到了9 个酚性化合物, 其中一个为新的糖基被酰化的酚性配糖体。用1D, 2D NMR 和MS 等现代波谱学方法鉴定为2-甲氧基-1 , 4-二苯酚-1-O - [6- O -(3-甲氧基-4-羟基苯甲酰基) ]-β-D-吡喃葡萄糖苷(1) 。8 个已知化合物分别为3′, 5′-二-C-β-D-吡喃葡萄糖基根皮素( 2)、芦丁(3) 、槲皮素-3- O-β-D-吡喃葡萄糖苷(4 ) 、邻苯三酚( 5 )、邻苯二酚( 6) 、对羟基苯甲酸( 7)、原儿茶酸(8) 和香草酸(9) 。化合物2 , 3 , 5 , 7～9 均为首次从草果果实中分离得到。     

Aerobic metabolism of 4-hydroxybenzoic acid in Archaea via an unusual pathway involving an intramolecular migration (NIH shift)

A novel haloarchaeal strain, Haloarcula sp. strain D1, grew aerobically on 4-hydroxybenzoic acid (4HBA) as a sole carbon and energy source and is the first member of the domain Archaea reported to do so. Unusually, D1 metabolized 4HBA via gentisic acid rather than via protocatechuic acid, hydroquinone, or catechol. Gentisate was detected in 4HBA-grown cultures, and gentisate 1,2-dioxygenase activity was induced in 4HBA-grown cells. Stoichiometric accumulation of gentisate from 4HBA was demonstrated in 4HBA-grown cell suspensions containing 2,2'-dipyridyl (which strongly inhibits gentisate 1,2-dioxygenase). To establish whether initial 1-hydroxylation of 4HBA with concomitant 1,2-carboxyl group migration to yield gentisate occurred, 2,6-dideutero-4HBA was synthesized and used as a substrate. Deuterated gentisate was recovered from cell suspensions and identified as 3-deutero-gentisate, using gas chromatography-mass spectrometry and proton nuclear magnetic resonance spectroscopy. This structural isomer would be expected only if a 1,2-carboxyl group migration had taken place, and it provides compelling evidence that the 4HBA pathway in Haloarcula sp. strain D1 involves a hydroxylation-induced intramolecular migration. To our knowledge, this is the first report of a pathway which involves such a transformation (called an NIH shift) in the domain ARCHAEA:     

Purification and Characterization of Catechol 1,2-Dioxygenase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. DS002 and Cloning,Sequencing of Partial <Emphasis Type="Italic">catA</Emphasis> Gene

Catechol 1,2-dioxygenase (C12O) was purified to electrophoretic homogeneity from Acinetobacter sp. DS002. The pure enzyme appears to be a homodimer with a molecular mass of 66 kDa. The apparent K_m and V_max for intradiol cleavage of catechol were 1.58 μM and 2 units per mg of protein respectively. Unlike other C12Os reported in the literature, the catechol 1,2-dioxygenase of Acinetobacter showed neither intradiol nor extradiol cleavage activity when substituted catechols were used as substrates. However, it has shown mild intradiol cleavage activity when benzenetriol was used as substrate. As determined by two dimensional electrophoresis (2DE) followed MALDI-TOF/TOF analyses and gel permeation chromatography, no isoforms of C12O was observed in Acinetobacter sp. DS002. Further, the C12O was seen only in cultures grown in benzoate and it was completely absent in succinate grown cultures. Based on the sequence information obtained from MS/MS data, degenerate primers were designed to amplify catA gene from the genomic DNA of Acinetobacter sp. DS002. The sequence of the PCR amplicon and deduced amino acid sequence showed 97% similarity with a catA gene of Acinetobacter baumannii AYE (YP_001713609).     

草果果实中的酚性成分

从草果（Amomum tsao-ko）果实的甲醇提取物中分离得到了9个酚性化合物,其中一个为新的糖基被酰化的酚性配糖体。用1D,2D NMR和MS等现代波谱学方法鉴定为2-甲氧基-1,4-二苯酚-1—0-[6-O-（3-甲氧基-4-羟基苯甲酰基）]-β-D-吡喃葡萄糖苷（1）。8个已知化合物分别为3’,5＇-二-C-β-D-吡喃葡萄糖基根皮素（2）、芦丁（3）、槲皮素-3-O-β-D-吡喃葡萄糖苷（4）、邻苯三酚（5）、邻苯二酚（6）、对羟基苯甲酸（7）、原儿茶酸（8）和香草酸（9）。化合物2,3,5,7—9均为首次从草果果实中分离得到。     

Phenolic Compound Utilization by the Soft Rot Fungus Lecythophora hoffmannii

Nine phenolic compounds were metabolized by the soft rot fungus Lecythophora hoffmannii via protocatechuic acid and subsequently cleaved by protocatechuate 3,4-dioxygenase as determined by oxygen uptake, substrate depletion, and ring cleavage analysis. Catechol was metabolized by catechol 1,2-dioxygenase. Fungal utilization of these aromatic compounds may be important in the metabolism of wood decay products.     

Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment

Aniline-degraders were isolated from activated sludge and environmental samples and classified into eight phylogenetic groups. Seven groups were classified into Gram-negative bacteria, such as Acidovorax sp., Acinetobacter sp., Delftia sp., Comamonas sp., and Pseudomonas sp., suggesting the possible dominance of Gram-negative aniline-degraders in the environment. Aniline degradative genes were cloned from D. acidovorans strain 7N, and the nucleotide sequence of the 8,039-bp fragment containing eight open reading frames was determined. Their deduced amino acid sequences showed homologies to glutamine synthetase (GS)-like protein, glutamine amidotransferase (GA)-like protein, large and small subunits of aniline dioxygenase, reductase, LysR-type regulator, small ferredoxin-like protein, and catechol 2,3-dioxygenase, suggesting a high similarity of this gene cluster to those in P. putida strain UCC22 and Acinetobacter sp. strain YAA. Polymerase chain reaction (PCR) and sequencing analyses of GS-like protein gene segments of other Gram-negative bacteria suggested that Gram-negative bacteria have aniline degradative gene that can be divided into two distinctive groups.     

Three types of phenol and p-cresol catabolism in phenol- and p-cresol-degrading bacteria isolated from river water continuously polluted with phenolic compounds

A total of 39 phenol- and p-cresol-degraders isolated from the river water continuously polluted with phenolic compounds of oil shale leachate were studied. Species identification by BIOLOG GN analysis revealed 21 strains of Pseudomonas fluorescens (4, 8 and 9 of biotypes A, C and G, respectively), 12 of Pseudomonas mendocina, four of Pseudomonas putida biotype A1, one of Pseudomonas corrugata and one of Acinetobacter genospecies 15. Computer-assisted analysis of rep-PCR fingerprints clustered the strains into groups with good concordance with the BIOLOG GN data. Three main catabolic types of degradation of phenol and p-cresol were revealed. Type I, or meta-meta type (15 strains), was characterized by meta cleavage of catechol by catechol 2,3-dioxygenase (C23O) during the growth on phenol and p-cresol. These strains carried C23O genes which gave PCR products with specific xylE-gene primers. Type II, or ortho-ortho type (13 strains), was characterized by the degradation of phenol through ortho fission of catechol by catechol 1,2-dioxygenase (C12O) and p-cresol via ortho cleavage of protocatechuic acid by protocatechuate 3,4-dioxygenase (PC34O). These strains carried phenol monooxygenase gene which gave PCR products with pheA-gene primers. Type III, or meta-ortho type (11 strains), was characterized by the degradation of phenol by C23O and p-cresol via the protocatechuate ortho pathway by the induction of PC34O and this carried C23O genes which gave PCR products with C23O-gene primers, but not with specific xylE-gene primers. In type III strains phenol also induced the p-cresol protocatechuate pathway, as revealed by the induction of p-cresol methylhydroxylase. These results demonstrate multiplicity of catabolic types of degradation of phenol and p-cresol and the existence of characteristic assemblages of species and specific genotypes among the strains isolated from the polluted river water.