Monocyclic aromatic hydrocarbon degradation by Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.     

Requirement of duplicated operons for maximal metabolism of phthalate by Rhodococcus sp. strain DK17

The operons encoding the transformation of phthalate to protocatechuate are duplicated and present on two different megaplasmids [pDK2 (330 kb) and pDK3 (750 kb)] in Rhodococcus sp. strain DK17. RT-PCR experiments using gene-specific primers showed that both the pDK2- and the pDK3-encoded dihydroxyphthalate decarboxylase genes are simultaneously expressed during growth on phthalate. The doubling time of the pDK2-cured mutant strain DK176 in minimal liquid medium with 5mM phthalate is 52.5% of that of the wild-type strain DK17. The data indicate that both copies of the phthalate operon are equally functional in DK17, and gene dosage is the main reason for slower growth of DK176 on phthalate.     

Benzylic and aryl hydroxylations of m-xylene by o-xylene dioxygenase from Rhodococcus sp. strain DK17

Escherichia coli cells expressing Rhodococcus DK17 o-xylene dioxygenase genes were used for bioconversion of m-xylene. Gas chromatography–mass spectrometry analysis of the oxidation products detected 3-methylbenzylalcohol and 2,4-dimethylphenol in the ratio 9:1. Molecular modeling suggests that o-xylene dioxygenase can hold xylene isomers at a kink region between α6 and α7 helices of the active site and α9 helix covers the substrates. m-Xylene is unlikely to locate at the active site with a methyl group facing the kink region because this configuration would not fit within the substrate-binding pocket. The m-xylene molecule can flip horizontally to expose the meta-position methyl group to the catalytic motif. In this configuration, 3-methylbenzylalcohol could be formed, presumably due to the meta effect. Alternatively, the m-xylene molecule can rotate counterclockwise, allowing the catalytic motif to hydroxylate at C-4 yielding 2,4-dimethylphenol. Site-directed mutagenesis combined with structural and functional analyses suggests that the alanine-218 and the aspartic acid-262 in the α7 and the α9 helices play an important role in positioning m-xylene, respectively.     

Monocyclic Aromatic Hydrocarbon Degradation by Rhodococcus sp. Strain DK17

Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.     

Differential degradation of bicyclics with aromatic and alicyclic rings by Rhodococcus sp. strain DK17

The metabolically versatile Rhodococcus sp. strain DK17 is able to grow on tetralin and indan but cannot use their respective desaturated counterparts, 1,2-dihydronaphthalene and indene, as sole carbon and energy sources. Metabolite analyses by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry clearly show that (i) the meta-cleavage dioxygenase mutant strain DK180 accumulates 5,6,7,8-tetrahydro-1,2-naphthalene diol, 1,2-indene diol, and 3,4-dihydro-naphthalene-1,2-diol from tetralin, indene, and 1,2-dihydronaphthalene, respectively, and (ii) when expressed in Escherichia coli, the DK17 o-xylene dioxygenase transforms tetralin, indene, and 1,2-dihydronaphthalene into tetralin cis-dihydrodiol, indan-1,2-diol, and cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, respectively. Tetralin, which is activated by aromatic hydroxylation, is degraded successfully via the ring cleavage pathway to support growth of DK17. Indene and 1,2-dihydronaphthalene do not serve as growth substrates because DK17 hydroxylates them on the alicyclic ring and further metabolism results in a dead-end metabolite. This study reveals that aromatic hydroxylation is a prerequisite for proper degradation of bicyclics with aromatic and alicyclic rings by DK17 and confirms the unique ability of the DK17 o-xylene dioxygenase to perform distinct regioselective hydroxylations.     

Identification of two-component regulatory genes involved in o-xylene degradation by Rhodococcus sp. strain DK17

Putative genes for a two-component signal transduction system (akbS and akbT) were detected near the alkylbenzene-degrading operon of Rhodococcus sp. DK17. Sequence analysis indicates that AkbS possesses potential ATP-binding and histidine autophosphorylation sites in the N- and C-terminal regions, respectively, and that AkbT has a typical response regulator domain. Mutant analysis combined with RT-PCR experiments further shows that AkbS is required to induce the expression of o-xylene dioxygenase in DK17.     

Functional characterization and molecular modeling of methylcatechol 2,3-dioxygenase from o-xylene-degrading Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 is known to metabolize o-xylene and toluene through the intermediates 3,4-dimethylcatechol and 3- and 4-methylcatechol, respectively, which are further cleaved by a common catechol 2,3-dioxygenase. A putative gene encoding this enzyme (akbC) was amplified by PCR, cloned, and expressed in Escherichia coli. Assessment of the enzyme activity expressed in E. coli combined with sequence analysis of a mutant gene demonstrated that the akbC gene encodes the bona fide catechol 2,3-dioxygenase (AkbC) for metabolism of o-xylene and alkylbenzenes such as toluene and ethylbenzene. Analysis of the deduced amino acid sequence indicates that AkbC consists of a new catechol 2,3-dioxygenase class specific for methyl-substituted catechols. A computer-aided molecular modeling studies suggest that amino acid residues (particularly Phe177) in the beta10-beta11 loop play an essential role in characterizing the substrate specificity of AkbC.     

Asymmetric oxidation of 1,3-propanediols to chiral hydroxyalkanoic acids by Rhodococcus sp. 2N

Rhodococcus sp. 2N was found as a 1,3-propanediols-oxidizing strain from soil samples through enrichment culture using 2,2-diethyl-1,3-propanediol (DEPD) as the sole carbon source. The culture condition of the strain 2N was optimized, and the highest activity was observed when 0.3% (w/v) DEPD was added in the culture medium as an inducer. Chiral HPLC analysis of the hydroxyalkanoic acid converted from 2-ethyl-2-methyl-1,3-propanediol (EMPD) revealed that the strain 2N catalyzed the (R)-selective oxidation of EMPD. The reaction products and intermediates from DEPD and EMPD were identified by nuclear magnetic resonance analyses, and the results suggested that only one hydroxymethyl group of the propanediols was converted to carboxy group via two oxidation steps. Under optimized conditions and after a 72-h reaction time, the strain 2N produced 28 mM (4.1 g/L) of 2-(hydroxymethyl)-2-methylbutanoic acid from EMPD with a molar conversion yield of 47% and 65% ee (R).     

红球菌DS—3脱除二苯并噻吩中有机硫的性能初探

从孤岛油田分离到一株红球菌(Rhodococcus sp.)DS—3,能专一地切断二苯并噻吩(DBT)中的C－S键,沿4S途径代谢,生成二羟联苯。实验证明,以2％的接种量脱除50μｇ／mL DBT底物中的硫效果最佳。在此条件下,适宜菌株生长和脱硫的碳源为葡萄糖,氯源为硝酸铵,初始PH为8．2,生长温度为30℃,15mmol/L的硫酸根离子能使其丧失脱硫能力。在上述适宜条件下,培养72h后DBT中34．04％的硫被脱除。     

Identification of functionally important amino acids in a novel indigo-producing oxygenase from <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. strain T104

A novel indigo-producing oxygenase gene, designated ipoA (1,197 bp) was characterized from Rhodococcus sp. strain T104. Three indigo-negative mutations (A58V, P59L, and G251D) were obtained through random mutagenesis using an E. coli mutator strain. Subsequent saturation mutagenesis resulted in the identification of nine and three amino acid substitutions that restore activity in the A58V and P59L mutants, respectively. Activity was not restored in the G251D mutation by any other amino acids. Interestingly, activity in the A58V mutant, where a methyl group is only replaced by an isopropyl side chain, is restored by a variety of amino acids, including polar ones. A molecular modeling study suggests that the residues at positions 58, 59, and 251 of the T104 IpoA enzyme are far from the active site, indicating that the mutations must alter the overall structure of the enzyme.     

Identification of a novel dioxygenase involved in metabolism of o-xylene, toluene, and ethylbenzene by Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 is able to grow on o-xylene, benzene, toluene, and ethylbenzene. DK17 harbors at least two megaplasmids, and the genes encoding the initial steps in alkylbenzene metabolism are present on the 330-kb pDK2. The genes encoding alkylbenzene degradation were cloned in a cosmid clone and sequenced completely to reveal 35 open reading frames (ORFs). Among the ORFs, we identified two nearly exact copies (one base difference) of genes encoding large and small subunits of an iron sulfur protein terminal oxygenase that are 6 kb apart from each other. Immediately downstream of one copy of the dioxygenase genes (akbA1a and akbA2a) is a gene encoding a dioxygenase ferredoxin component (akbA3), and downstream of the other copy (akbA1b and akbA2b) are genes putatively encoding a meta-cleavage pathway. RT-PCR experiments show that the two copies of the dioxygenase genes are operonic with the downstream putative catabolic genes and that both operons are induced by o-xylene. When expressed in Escherichia coli, AkbA1a-AkbA2a-AkbA3 transformed o-xylene into 2,3- and 3,4-dimethylphenol. These were apparently derived from an unstable o-xylene cis-3,4-dihydrodiol, which readily dehydrates. This indicates a single point of attack of the dioxygenase on the aromatic ring. In contrast, attack of AkbA1a-AkbA2a-AkbA3 on ethylbenzene resulted in the formation of two different cis-dihydrodiols resulting from an oxidation at the 2,3 and the 3,4 positions on the aromatic ring, respectively.     

Isolation and identification of berberine and berberrubine metabolites by berberine-utilizing bacterium Rhodococcus sp. strain BD7100

Based on the finding of a novel berberine (BBR)-utilizing bacterium, Rhodococcus sp. strain BD7100, we investigated the degradation of BBR and its analog berberrubine (BRU). Resting cells of BD7100 demethylenated BBR and BRU, yielding benzeneacetic acid analogs. Isolation of benzeneacetic acid analogs suggested that BD7100 degraded the isoquinoline ring of the protoberberine skeleton. This work represents the first report of cleavage of protoberberine skeleton by a microorganism.     

Inhibition of diethyl ether degradation in Rhodococcus sp. strain DEE5151 by glutaraldehyde and ethyl vinyl ether

Alkyl ether-degrading Rhodococcus sp. strain DEE5151, isolated from activated sewage sludge, has an activity for the oxidation of a variety of alkyl ethers, aralkyl ethers and dibenzyl ether. The whole cell activity for diethyl ether oxidation was effectively inhibited by 2,3-dihydrofurane, ethyl vinyl ether and glutaraldehyde. Glutaraldehyde of less than 30 microM inhibited the activity by a competitive manner with the inhibition constant, K(I) of 7.07+/-1.36 microM. The inhibition type became mixed at higher glutaraldehyde concentrations >30 microM, probably due to the inactivation of the cell activity by the Schiff-base formation. Structurally analogous ethyl vinyl ether inhibited the diethyl ether oxidation activity in a mixed manner with decreasing the apparent maximum oxidation rate, v(max)(app), and increasing the apparent Michaelis-Menten constant, K(M)(app). The mixed type inhibition by ethyl vinyl ether seemed to be introduced not only by the structure similarity with diethyl ether, but also by the reactivity of the vinyl ether with cellular components in the whole cell system.     

Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil

Aims:  The aim of this study is to isolate and characterize organisms capable of utilizing high concentration atrazine from the contaminated sites.
Methods and Results:  A selective enrichment was used for isolating atrazine-degrading organisms from the contaminated sites resulting in isolation of an efficient atrazine-degrading organism designated as strain MB-P1. On the basis of 16S rRNA gene sequencing, total cellular fatty acid analysis and physiological and biochemical tests, strain MB-P1 was identified as a member of genus Rhodococcus . High performance liquid chromatography was performed to identify the atrazine degradation intermediates demonstrating that the degradation proceeds via formation of 'de-ethylatrazine' and 'de-isopropylatrazine'. Further, plasmid curing by SDS method showed atrazine-degrading gene(s) to be plasmid-encoded.
Conclusions:  We have successfully isolated a Rhodococcus sp. strain MB-P1 which is capable of utilizing atrazine as sole source of carbon and energy at very high concentrations of 1000 ppm. The pathway for degradation of atrazine has also been determined. The metabolic gene(s) responsible for atrazine degradation was found to be plasmid-encoded.
Significance and Impact of the Study:  Rhodococcus sp. strain MB-P1 could be used as an ideal model system for in-situ degradation and restoration of ecological niches which are heavily contaminated with atrazine.     

Degradation of benzene by a Rhodococcus sp. using immobilized cell systems

The continuous degradation of benzene by a Rhodococcus sp. using free and immobilized cell systems was compared. Cell entrapment in calcium and strontium alginate beads and adhesion on support materials such as glass beads were found to be unsatisfactory. Degradation of benzene by cells immobilized in either ceramic or cellulose carriers proved to be more efficient than its non-immobilized counterpart. A retention time of 36 h was required to effect a 97% degradation of benzene using suspended free cells while cells immobilized on cellulose or ceramic carriers effected 97% degradation at 24 and 18 h, respectively. Recycling of the ceramic carriers was also possible and resulted in an even shorter retention time of 12h to effect a 97% degradation of benzene. Cell adhesion on the support materials was confirmed by scanning electron microscopy.     

红球菌HX-2所产胞外多糖的特性和对Cu2+的吸附

【背景】目前,微生物所产胞外多糖(exopolysaccharide,EPS)的理化性质及其在重金属吸附中的应用受到了广泛关注。【目的】研究红球菌HX-2所产胞外多糖的理化性质,并探究其对重金属的吸附情况。【方法】使用离子交换和凝胶色谱分离法对胞外多糖粗品进行纯化;利用苯酚硫酸法测胞外多糖中糖含量;用Bradford试剂盒检测胞外多糖中蛋白含量;使用甲醇萃取法检测胞外多糖中脂质含量;用高效液相色谱(high performance liquid chromatography,HPLC)法分析胞外多糖中单糖组成;用扫描电镜(scanningelectronmicroscopy,SEM)法观察多糖表面形态;通过等温吸附模型和动力学模型探究胞外多糖对重金属的吸附效果。【结果】测得胞外多糖主要成分EPS-G-1中总糖含量为78.43%,蛋白含量为8.31%,脂质含量为8.22%;纯化后胞外多糖中单糖组成为葡萄糖、甘露糖、半乳糖、葡萄糖醛酸和岩藻糖,质量比为27.31:26.67:24.83:15.85:4.80;通过等温吸附模型拟合得到HX-2所产胞外多糖对Cu~(2+)的最大吸附量为144.93 mg/g。【结论】红球菌HX-2所产胞外多糖对水体中Cu~(2+)具有良好的吸附作用,可用于工业废水中重金属离子的处理。