Aerobic metabolism of phenylacetic acids in Azoarcus evansii

The aerobic metabolism of phenylacetic acid (PA) and 4-hydroxyphenylacetic acid (4-OHPA) was investigated in the beta-proteobacterium Azoarcus evansii. Evidence for the existence of two independent catabolic pathways for PA and 4-OHPA is presented. 4-OHPA metabolism involves the formation of 2,5-dihydroxyphenylacetate (homogentisate) and maleylacetoacetate catalyzed by specifically induced 4-OHPA 1-monooxygenase and homogentisate 1,2-dioxygenase. The metabolism of PA starts by its activation to phenylacetyl-CoA (PA-CoA) via an aerobically induced phenylacetate-coenzyme A ligase. Phenylalanine (Phe) aerobic metabolism in this bacterium proceeds also via PA and PA-CoA. Whole cells of A. evansii transformed [1-(14)C]PA to (14)C-phenylacetyl-CoA and subsequently to a number of unknown labeled products, which were also observed in PA-degrading bacteria from different phylogenetic groups, i.e. Escherichia coli, Rhodopseudomonas palustrisand Bacillus stearothermophilus. A chromosomal region from A. evansiiof 11.5 kb containing a cluster of 11 phenylacetic acid catabolic ( paa) genes ( paaYZGHIKABCDE) was sequenced and characterized. The derived gene products were similar to the characterized putative gene products involved in PA catabolism in E. coli and Pseudomonas putida and to other putative PA catabolic gene products of diverse bacteria. RT-PCR analysis of the paa genes of A. evansiigrowing aerobically with PA showed a probable organization of the paa genes in three operons. The similarity of the PA metabolic products pattern and of gene sequences suggests a common aerobic bacterial PA pathway.     

First report of HGD mutations in a Chinese with alkaptonuria

Alkaptonuria (AKU) is one of the first prototypic inborn errors in metabolism and the first human disease found to be transmitted via Mendelian autosomal recessive inheritance. It is caused by HGD mutations, which leads to a deficiency in homogentisate 1,2-dioxygenase (HGD) activity.     

Loss of Homogentisate 1,2-Dioxygenase Activity in Bacillus anthracis Results in Accumulation of Protective Pigment

Melanin production is important to the pathogenicity and survival of some bacterial pathogens. In Bacillus anthracis, loss of hmgA, encoding homogentisate 1,2-dioxygenase, results in accumulation of a melanin-like pigment called pyomelanin. Pyomelanin is produced in the mutant as a byproduct of disrupted catabolism of L-tyrosine and L-phenylalanine. Accumulation of pyomelanin protects B. anthracis cells from UV damage but not from oxidative damage. Neither loss of hmgA nor accumulation of pyomelanin alter virulence gene expression, sporulation or germination. This is the first investigation of homogentisate 1,2-dioxygenase activity in the Gram-positive bacteria, and these results provide insight into a conserved aspect of bacterial physiology.     

Isolation and characterization of polycyclic aromatic hydrocarbons-degrading Sphingomonas sp. strain ZL5

A bacterial strain ZL5, capable of growing on phenanthrene as a sole carbon and energy source but not naphthalene, was isolated by selective enrichment from crude-oil-contaminated soil of Liaohe Oil Field in China. The isolate was identified as a Sphingomonas sp. strain on the basis of 16S ribosomal DNA analysis. Strain ZL5 grown on phenanthrene exhibited catechol 2,3-dioxygenase (C23O) activity but no catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities. This suggests that the mode of cleavage of phenanthrene by strain ZL5 could be meta via the intermediate catechol, which is different from the protocatechuate way of other two bacteria, Alcaligenes faecelis AFK2 and Nocardioides sp. strain KP7, also capable of growing on phenanthrene but not naphthalene. A resident plasmid (approximately 60 kb in size), designated as pZL, was detected from strain ZL5. Curing the plasmid with mitomycin C and transferring the plasmid to E. coli revealed that pZL was responsible for polycyclic aromatic hydrocarbons degradation. The C23O gene located on plasmid pZL was cloned and overexpressed in E. coli JM109(DE3). The ring-fission activity of the purified C23O from the recombinant E. coli on dihydroxylated aromatics was in order of catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol > 3,4-dihydroxyphenanthrene > 3-chlorocatechol.     

Metabolism of tetralin (1,2,3,4-tetrahydronaphthalene) in Corynebacterium sp. strain C125.

Corynebacterium sp. strain C125, originally isolated on o-xylene, was selected for its ability to grow on tetralin (1,2,3,4-tetrahydronaphthalene) as the sole source of carbon and energy. The catabolism of tetralin in Corynebacterium sp. strain C125 was shown to proceed via initial hydroxylation of the benzene nucleus at positions C-5 and C-6, resulting in the formation of the corresponding cis-dihydro diol. Subsequently, the dihydro diol was dehydrogenated by a NAD-dependent dehydrogenase to 5,6,7,8-tetrahydro-1,2-naphthalene diol. The aromatic ring was cleaved in the extradiol position by a catechol-2,3-dioxygenase. The ring fission product was subject to a hydrolytic attack, resulting in the formation of a carboxylic acid-substituted cyclohexanone. This is the first report of the catabolism of tetralin via degradation of the aromatic moiety.     

Purification and properties of gentisate 1,2-dioxygenase from Moraxella osloensis.

Gentisate:oxygen 1,2-oxidoreductase (decyclizing) (EC 1.13.11.4; gentisate 1,2-dioxygenase) from Moraxella osloensis was purified to homogeneity as shown by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of about 154,000 and gives rise to subunits of molecular weight 40,000 in the presence of sodium dodecyl sulfate. Gentisate 1,2-dioxygenase showed broad substrate specificity and attacked a range of halogen- and alkyl-substituted gentisic acids. Maleylpyruvate, the product formed from gentisate, was degraded by cell extracts supplemented with reduced glutathione, but substituted maleylpyruvates were not attacked under these conditions.     

Simultaneous biodegradation of chlorobenzene and toluene by a Pseudomonas strain.

Pseudomonas sp. strain JS6 grows on a wide range of chloro- and methylaromatic substrates. The simultaneous degradation of these compounds is prevented in most previously studied isolates because the catabolic pathways are incompatible. The purpose of this study was to determine whether strain JS6 could degrade mixtures of chloro- and methyl-substituted aromatic compounds. Strain JS6 was maintained in a chemostat on a minimal medium with toluene or chlorobenzene as the sole carbon source, supplied via a syringe pump. Strain JS6 contained an active catechol 2,3-dioxygenase when grown in the presence of chloroaromatic compounds; however, in cell extracts, this enzyme was strongly inhibited by 3-chlorocatechol. When cells grown to steady state on toluene were exposed to 50% toluene-50% chlorobenzene, 3-chlorocatechol and 3-methylcatechol accumulated in the medium and the cell density decreased. After 3 h, the enzyme activities of the modified ortho ring fission pathway were induced, the metabolites disappeared, and the cell density returned to previous levels. In cell extracts, 3-methylcatechol was degraded by both catechol 1,2- and catechol 2,3-dioxygenase. Strain JS62, a catechol 2,3-dioxygenase mutant of JS6, grew on toluene, and ring cleavage of 3-methylcatechol was catalyzed by catechol 1,2-dioxygenase. The transient metabolite 2-methyllactone was identified in chlorobenzene-grown JS6 cultures exposed to toluene. These results indicate that strain JS6 can degrade mixtures of chloro- and methylaromatic compounds by means of a modified ortho ring fission pathway.     

Simultaneous biodegradation of chlorobenzene and toluene by a Pseudomonas strain

Pseudomonas sp. strain JS6 grows on a wide range of chloro- and methylaromatic substrates. The simultaneous degradation of these compounds is prevented in most previously studied isolates because the catabolic pathways are incompatible. The purpose of this study was to determine whether strain JS6 could degrade mixtures of chloro- and methyl-substituted aromatic compounds. Strain JS6 was maintained in a chemostat on a minimal medium with toluene or chlorobenzene as the sole carbon source, supplied via a syringe pump. Strain JS6 contained an active catechol 2,3-dioxygenase when grown in the presence of chloroaromatic compounds; however, in cell extracts, this enzyme was strongly inhibited by 3-chlorocatechol. When cells grown to steady state on toluene were exposed to 50% toluene-50% chlorobenzene, 3-chlorocatechol and 3-methylcatechol accumulated in the medium and the cell density decreased. After 3 h, the enzyme activities of the modified ortho ring fission pathway were induced, the metabolites disappeared, and the cell density returned to previous levels. In cell extracts, 3-methylcatechol was degraded by both catechol 1,2- and catechol 2,3-dioxygenase. Strain JS62, a catechol 2,3-dioxygenase mutant of JS6, grew on toluene, and ring cleavage of 3-methylcatechol was catalyzed by catechol 1,2-dioxygenase. The transient metabolite 2-methyllactone was identified in chlorobenzene-grown JS6 cultures exposed to toluene. These results indicate that strain JS6 can degrade mixtures of chloro- and methylaromatic compounds by means of a modified ortho ring fission pathway.     

Degradation of guaiacol glyceryl ether (GGE) by Bacillus subtilis

Summary Bacillus subtilis utilized guaiacol glyceryl ether (GGE) as sole carbon source and catabolized it via guaiacol and catechol. Cell free extracts of GGE grown cells contained high levels of catechol 1,2-dioxygenase and cleaved catechol via the ortho pathway. Nutrients such as sugars, organic acids, methanol, nitrogen and phosphate influenced the catabolism of GGE by B. subtilis.     

Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp

Eight actinomycetes of the genera Amycolatopsis and Streptomyces were tested for the degradation of aromatic compounds by growth in a liquid medium containing benzoate, monohydroxylated benzoates, or quinate as the principal carbon source. Benzoate was converted to catechol. The key intermediate in the degradation of salicylate was either catechol or gentisate, while m-hydroxybenzoate was metabolized via gentisate or protocatechuate. p-Hydroxybenzoate and quinate were converted to protocatechuate. Catechol, gentisate, and protocatechuate were cleaved by catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, and protocatechuate 3,4-dioxygenase, respectively. The requirement for glutathione in the gentisate pathway was dependent on the substrate and the particular strain. The conversion of p-hydroxybenzoate to protocatechuate by p-hydroxybenzoate hydroxylase was gratuitously induced by all substrates that were metabolized via protocatechuate as an intermediate, while protocatechuate 3,4-dioxygenase was gratuitously induced by benzoate and salicylate in two Amycolatopsis strains.     

Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp.

Eight actinomycetes of the genera Amycolatopsis and Streptomyces were tested for the degradation of aromatic compounds by growth in a liquid medium containing benzoate, monohydroxylated benzoates, or quinate as the principal carbon source. Benzoate was converted to catechol. The key intermediate in the degradation of salicylate was either catechol or gentisate, while m-hydroxybenzoate was metabolized via gentisate or protocatechuate. p-Hydroxybenzoate and quinate were converted to protocatechuate. Catechol, gentisate, and protocatechuate were cleaved by catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, and protocatechuate 3,4-dioxygenase, respectively. The requirement for glutathione in the gentisate pathway was dependent on the substrate and the particular strain. The conversion of p-hydroxybenzoate to protocatechuate by p-hydroxybenzoate hydroxylase was gratuitously induced by all substrates that were metabolized via protocatechuate as an intermediate, while protocatechuate 3,4-dioxygenase was gratuitously induced by benzoate and salicylate in two Amycolatopsis strains.     

Degradation of homovanillate by a strain of Variovorax paradoxus via ring hydroxylation

Abstract A newly isolated strain of Variovorax paradoxus could grow on homovanillate and several monohydroxylated phenylacetic acids. During growth on homovanillate, the organism formed separate NAD(P)H-dependent hydroxylases with activity towards 4-hydroxyphenylacetic acid and homovanillate. Homovanillate hydroxylase catalysed a typical monooxygenase reaction and had little activity towards 4-hydroxyphenylacetic acid. GC-MS and TLC analysis suggested that homovanillate was 1-hydroxylated to yield a dihydroxymonomethoxyphenylacetic acid which served as a substrate for homogentisate 1,2-dioxygenase. Methanol, but not formaldehyde, was released either during ring-cleavage or subsequent metabolism of the ring-cleavage product.     

Molecular characterization of a novel ortho-nitrophenol catabolic gene cluster in Alcaligenes sp. strain NyZ215

Alcaligenes sp. strain NyZ215 was isolated for its ability to grow on ortho-nitrophenol (ONP) as the sole source of carbon, nitrogen, and energy and was shown to degrade ONP via a catechol ortho-cleavage pathway. A 10,152-bp DNA fragment extending from a conserved region of the catechol 1,2-dioxygenase gene was obtained by genome walking. Of seven complete open reading frames deduced from this fragment, three (onpABC) have been shown to encode the enzymes involved in the initial reactions of ONP catabolism in this strain. OnpA, which shares 26% identity with salicylate 1-monooxygenase of Pseudomonas stutzeri AN10, is an ONP 2-monooxygenase (EC 1.14.13.31) which converts ONP to catechol in the presence of NADPH, with concomitant nitrite release. OnpC is a catechol 1,2-dioxygenase catalyzing the oxidation of catechol to cis,cis-muconic acid. OnpB exhibits 54% identity with the reductase subunit of vanillate O-demethylase in Pseudomonas fluorescens BF13. OnpAB (but not OnpA alone) conferred on the catechol utilizer Pseudomonas putida PaW340 the ability to grow on ONP. This suggests that OnpB may also be involved in ONP degradation in vivo as an o-benzoquinone reductase converting o-benzoquinone to catechol. This is analogous to the reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone by a tetrachlorobenzoquinone reductase (PcpD, 38% identity with OnpB) in the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723.     

好氧氯苯降解菌的分离鉴定

【目的】分离好氧氯苯降解菌,并通过研究降解特性为应用提供理论依据。【方法】利用富集培养技术分离菌株,通过形态、生理生化反应特征及16S rRNA基因序列分析鉴定菌株,测定培养液中氯苯、其它氯苯类化合物和氯离子的浓度以及菌体细胞的密度和菌体细胞粗提液中邻苯二酚双加氧酶的活性,研究菌株的降解特性。【结果】16S rRNA基因序列相似性比较表明,分离出的菌株与乙酸钙不动杆菌(Acinetobacter calcoaceticus)的相似性高达98.5%。以初始浓度为50mg/L的氯苯为唯一碳源和能源时,120h内菌株对氯苯的降解率高达98.2%,氯离子净释放量和氯苯降解量的摩尔比范围为1:1.85-1:1.39,菌体细胞粗提液中邻苯二酚1,2-双加氧酶的平均活性为0.538U/mg蛋白质。加入葡萄糖后,菌体细胞数量和氯离子浓度明显增加,但单位细胞的氯苯降解能力明显下降。在二氯苯和三氯苯共存时,菌株对氯苯的降解能力受到明显的抑制作用,但对二氯苯有一定的降解作用,降解能力大小顺序为:1,3-二氯苯1,2-二氯苯1,4-二氯苯。【结论】分离出的好氧氯苯降解菌属于Acinetobacter属菌株,该菌株对氯苯和二氯苯均具有降解作用,可能通过邻位裂环途径降解氯苯,氯苯对菌株的降解能力和邻苯二酚1,2-双加氧酶的活性具有明显的增强作用。     

假单胞菌DLL-E4尿黑酸降解基因的克隆与功能的初步分析

通过接合转移将质粒pSC123上的转座子Tn5随机插入到DLL-E4基因组DNA中,从大约8,000个突变子中筛选到1株在LB培养基上积累红褐色物质的突变株M18,该突变株不能以L-苯丙氨酸(L-Phenylalanine, Phe)为唯一碳源生长。SEFA-PCR扩增转座子侧翼序列发现其与已报道的尿黑酸1,2-双加氧酶基因hmgA的同源性为92%。将hmgA定向克隆至表达载体pET-29a中,转化至Escherichia coli BL21,经IPTG诱导后可表达分子量约为48kD的蛋白;诱导后转化子粗酶液对尿黑酸有很好的降解效果。将hmgA连入自杀性载体pEX19Gm,通过同源重组整合至M18染色体中,使其恢复了DLL-E4利用Phe的能力,证实了HmgA是尿黑酸苯环裂解酶。