假单胞菌ZWL73降解4-氯硝基苯的代谢途径研究

氯代硝基芳香烃是一类环境中难以降解的有毒污染物。一株高效分解4-氯硝基苯的假单胞菌分离于4-氯硝基苯污染土壤, 可以完全降解4-氯硝基苯, 并以之为C源、N源生长。为阐明其降解4-氯硝基苯的代谢途径, 通过对以底物生长的降解菌的酶学分析, 检测到其还原降解的两个关键酶即初始酶硝基还原酶和苯环开环酶2-氨基-5-氯酚1, 6-双加氧酶的活性; 结合其它检测如培养液中降解产物分析、相关底物生长实验结果, 确定了其降解途径是通过部分还原途径。     

一株降解对氯硝基苯的Comamonas sp.CNB1的分离鉴定及其降解特性

从处理某化工厂污水的活性污泥中分离到一株降解对氯硝基苯的细菌CNB1菌株。经过对其形态特征、生理生化、以及16S rDNA序列分析,该菌株初步鉴定为Comamonas sp.,进一步研究表明,该菌株能够以对氯硝基苯为唯一碳源、氮源和能源生长。生长过程中,氯离子释放同步于对氯硝基苯降解,且氯离子的释放量与对氯硝基苯的降解量相当。该细菌利用对氯硝基苯生长的最适生长温度和pH分别为28℃和9.0。测定了降解途径中相关酶的活性,表明初始降解过程是由对氯硝基苯还原酶催化的硝基还原反应,芳环的裂解是由2-氨基苯酚1,6-双加氧酶催化。     

一株降解对氯硝基苯的Comamonas sp. CNB1的分离鉴定及其降解特性

从处理某化工厂污水的活性污泥中分离到一株降解对氯硝基苯的细菌CNB1菌株。经过对其形态特征、生理生化、以及16S rDNA序列分析,该菌株初步鉴定为Comamonas sp.,进一步研究表明,该菌株能够以对氯硝基苯为唯一碳源、氮源和能源生长。生长过程中,氯离子释放同步于对氯硝基苯降解,且氯离子的释放量与对氯硝基苯的降解量相当。该细菌利用对氯硝基苯生长的最适生长温度和pH分别为28℃和9.0。测定了降解途径中相关酶的活性,表明初始降解过程是由对氯硝基苯还原酶催化的硝基还原反应,芳环的裂解是由2_氨基苯酚1, 6双加氧酶催化。     

盐胁迫条件下古菌超氧化物歧化酶增强细菌降解硝基酚

【背景】Burkholderia sp. SJ98利用对硝基酚和2-氯-4-硝基酚为唯一碳源和能源进行生长,通过异源表达嗜盐古菌Haloferax sp. D1227中的超氧化物歧化酶SodA,使菌株SJ98在500 mmol/L NaCl条件下仍具有降解对硝基酚的能力。然而该重组细菌在普通和高盐条件下其降解基因的转录和降解酶比活力的高低,以及该菌在高盐条件下是否还能降解对硝基酚衍生物尚未知晓。【目的】研究Burkholderia sp. SJ98的耐盐上限,观察含有sodA的细菌SJ98在普通和高盐条件下降解对硝基酚和2-氯-4-硝基酚的能力,检测重组菌中pnpA基因的转录和硝基酚单加氧酶的活力。【方法】在添加葡萄糖、对硝基酚或2-氯-4-硝基酚的无机盐培养基(分别含400-800 mmol/L NaCl)或M9培养基(含0和500 mmol/L NaCl)中培养细菌SJ98及其重组菌。通过紫外分光光度计和高效液相色谱法检测菌株生长和底物降解。通过实时荧光定量PCR分别以两种硝基酚为诱导物,检测未添加和添加500 mmol/L NaCl时,硝基酚单加氧酶编码基因pnpA的转录量变化。利用紫外分光光度计分别以两种硝基酚为底物,检测在添加500 mmol/L NaCl时,重组菌和空载体菌的粗酶液中硝基酚单加氧酶对两种底物的活力变化。【结果】野生型菌株SJ98以葡萄糖为碳源生长的NaCl耐受浓度是600mmol/L。未添加NaCl时,重组菌SJ98[pCM-pnpR-PpnpA-sodA-rfp]生长和降解对硝基酚的能力远优于野生菌。添加500 mmol/L NaCl时,重组菌SJ98[pBBR-sodA]仍保持了利用2-氯-4-硝基苯酚底物生长和降解该底物的能力,而空载体菌SJ98[pBBR1MCS-2]的生长和降解能力完全丧失;重组菌SJ98[pBBR-sodA]粗酶液中单加氧酶对于对硝基酚和2-氯-4-硝基酚的活力均约为野生菌的1/3。分别以两种硝基酚为诱导物时,无论是否添加NaCl,重组菌SJ98[pBBR-sodA]中硝基酚单加氧酶编码基因pnpA的转录量比野生型中高出约17-25倍;但添加500 mmol/L NaCl时,pnpA的转录均受到部分抑制。【结论】本研究为利用古菌超氧化物歧化酶对细菌进行改造以提高普通环境和高盐环境中细菌降解硝基芳烃污染物能力的应用提供了潜在的可行性。     

硝基苯污染物的生物降解途径

硝基苯是一种有毒化合物,目前,关于硝基苯污染物的生物降解已进行了大量的研究。综述了生物降解硝基苯的两种主要途径氧化途径和部分还原途径,介绍了两种途径降解硝基苯的具体机制及相关酶和编码基因的特点,并对两种降解途径进行了简要的对比分析,为硝基苯及其它有机污染物生物降解技术的开发应用提供依据。     

探索假单胞菌降解4-氯硝基苯代谢新途径

尽管硝基芳香烃的微生物降解机理已经研究了二十多年,但是还有较多的硝基化毒性化合物的生物学降解机理尚待探索,特别是氯代硝基芳香烃芳环类化合物不易被活化,从而比单取代芳香烃更难以降解.     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。     

厌氧微生物菌群XH-1对2,4,6-三氯苯酚的降解特性

有机污染物2,4,6-三氯苯酚(2,4,6-TCP)普遍存在于地下水和河流底泥等厌氧环境中。为了探究厌氧微生物菌群XH-1对2,4,6-TCP的降解能力,本研究以2,4,6-TCP为底物,接种XH-1建立微宇宙培养体系,并以中间产物4-氯苯酚(4-CP)和苯酚为底物分别进行分段富集培养,利用高效液相色谱分析底物的降解转化,同时基于16S rRNA基因高通量测序分析微生物群落结构变化。结果表明: 2,4,6-TCP(122 μmol·L^-1)以0.15 μmol·d^-1的速率在80 d内被完全降解转化,降解中间产物分别为2,4-二氯苯酚(2,4-DCP)、4-氯苯酚和苯酚,所有中间产物最终在325 d被完全降解。高通量测序结果表明,脱卤杆菌和脱卤球菌可能驱动2,4,6-TCP还原脱氯,其中,脱卤球菌可能在4-CP的脱氯转化中发挥重要作用,并与丁酸互营菌和产甲烷菌联合作用彻底降解2,4,6-TCP。     

三氯卡班降解菌株Sphingomonas sp. YL-JM2C中多个邻苯二酚双加氧酶的功能

【目的】研究Sphingomonas sp.YL-JM2C菌株的生长特性,确定以三氯卡班作为碳源的生长情况。挖掘菌株YL-JM2C潜在的邻苯二酚1,2-双加氧酶及邻苯二酚2,3-双加氧酶基因,在大肠杆菌(Escherichia coli)中异源表达邻苯二酚双加氧酶基因并研究其酶学性质。【方法】优化S.sp.YL-JM2C菌株以三氯卡班作为碳源时的培养条件,并利用全自动生长曲线测定仪测定菌株生长情况,绘制生长曲线。通过生物信息学方法挖掘潜在的邻苯二酚双加氧酶基因,并分别在Escherichia coli BL21(DE3)中进行异源表达,通过AKTA快速纯化系统纯化蛋白,分别以邻苯二酚、3-和4-氯邻苯二酚为底物检测重组蛋白的酶学特性。【结果】菌株在pH为7.0-7.5时生长最优。在以浓度为4-8 mg/L的三氯卡班做为底物时,菌株适宜生长。当R2A培养基仅含有0.01%酵母提取物和无机盐时,加入终浓度为4 mg/L的三氯卡班可促进菌株生长。挖掘到6个潜在的邻苯二酚双加氧酶基因stcA1、stcA2、stcA3、stcE1、stcE2和stcE3,表达并通过粗酶液分析证明其中5个基因stcA1、stcA2、stcA3、stcE1和stcE2编码的酶均具有邻苯二酚双加氧酶和氯邻苯二酚双加氧酶的活性;纯化酶的底物范围研究揭示了StcA1、StcA2和StcA3均属于Ⅱ型邻苯二酚1,2-双加氧酶,StcE1和StcE2为两个新型邻苯二酚2,3-双加氧酶;它们酶动力学分析研究证明了5个酶对邻苯二酚的亲和力和催化效率最高,4-氯邻苯二酚次之。【结论】在同一菌株中发现了5个具有功能的邻苯二酚双加氧酶基因,stcA1、stcA2和stcA3编码的酶均属于Ⅱ型邻苯二酚1,2-双加氧酶,stcE1和stcE2为两个新型邻苯二酚2,3-双加氧酶编码基因。5个酶均具有催化邻苯二酚和氯邻苯二酚开环反应的功能,这为更好地理解微生物基因组内代谢邻苯二酚及其衍生物氯代邻苯二酚基因的多样性奠定了基础。     

酿酒酵母B5不对称还原制备手性药物中间体R-2′-氯-1-苯乙醇的研究

从11株微生物中筛选出4株具有不对称还原2′-氯-苯乙酮能力的酵母,其中酿酒酵母B5的还原产率与对映体选择性最佳。确定了酿酒酵母B5对2′-氯-苯乙酮还原的最佳反应时间为24h;最佳pH 8.0;最佳反应温度为25℃;最佳共底物为5%（体积比）乙醇。同时研究了底物浓度、微生物的量、微生物的培养条件等对反应产率和立体选择性的影响。细胞浓度为10.75mg/mL（细胞干重/反应体积）的酿酒酵母B5可将647mmol/L的2′-氯-苯乙酮100%地转化为R-2′-氯-1-苯乙醇,其对映体选择性为100%。酿酒酵母B5可重复利用的特点可提高产物的产量。     

Characterization of genes involved in the initial reactions of 4-chloronitrobenzene degradation in Pseudomonas putida ZWL73

The genes encoding enzymes involved in the initial reactions during degradation of 4-chloronitrobenzene (4CNB) were characterized from the 4CNB utilizer Pseudomonas putida ZWL73, in which a partial reductive pathway was adopted. A DNA fragment containing genes coding for chloronitrobenzene nitroreductase (CnbA) and hydroxylaminobenzene mutase (CnbB) were PCR-amplified and subsequently sequenced. These two genes were actively expressed in Escherichia coli, and recombinant E. coli cells catalyzed the conversion of 4CNB to 2-amino-5-chlorophenol, which is the ring-cleavage substrate in the degradation of 4CNB. Phylogenetic analyses on sequences of chloronitrobenzene nitroreductase and hydroxylaminobenzene mutase revealed that these two enzymes are closely related to the functionally identified nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas strains JS45 and HS12. The nitroreductase from strain ZWL73 showed a higher specific activity toward 4CNB than nitrobenzene (approximately at a ratio of 1.6:1 for the recombinant or 2:1 for the wild type), which is in contrast to the case where the nitroreductase from nitrobenzene utilizers Pseudomonas pseudoalcaligenes JS45 with an apparently lower specific activity against 4CNB than nitrobenzene (0.16:1) [Kadiyala et al. Appl Environ Microbiol 69:6520–6526, 2003]. This suggests that the nitroreductase from 4-chloronitrobenzene utilizer P. putida ZWL73 may have evolved to prefer chloronitrobenzene to nitrobenzene as its substrate.Y.X. and J.-F.W. equally contributed to this work.     

Proteomic and molecular investigation on the physiological adaptation of Comamonas sp. strain CNB-1 growing on 4-chloronitrobenzene

Comamonas sp. strain CNB-1 can utilize 4-chloronitrobenzene (4CNB) as sole carbon and nitrogen source for growth. Previous studies were focused on 4CNB degradative pathway and have showed that CNB-1 contained a plasmid pCNB1 harboring the genes (cnbABCaCbDEFGH, cnbZ) for the enzymes involving in 4CNB degradation, but only three gene products (CnbCa, CnbCb, and CnbZ) were identified in CNB-1 cells. Comamonas strain CNB-2 that lost pCNB1 was not able to grow on 4CNB. In this study, physiological adaptation to 4CNB by CNB-1 was investigated with proteomic and molecular tools. Comparative proteomes of strains CNB-1 and CNB-2 grown on 4CNB and/or succinate revealed that adaptation to 4CNB by CNB-1 included specific degradative pathway and general physiological responses: (1) Seven gene products (CnbA, CnbCa, CnbCb, CnbD, CnbE, CnbF, and CnbZ) for 4CNB degradation were identified in 4CNB-grown cells, and they were constitutively synthesized in CNB-1. Two genes cnbE and cnbF were cloned and simultaneously expressed in E. coli. The CnbE and CnbF together catalyzed the conversion of 2-oxohex-4-ene-5-chloro-1,6-dioate into 2-oxo-4-hydroxy-5-chloro-valeric acid; (2) Enzymes involving in glycolysis, tricarboxylic acid cycle, and synthesis of glutamate increased their abundances in 4CNB-grown cells.     

Patchwork assembly of nag-like nitroarene dioxygenase genes and the 3-chlorocatechol degradation cluster for evolution of the 2-chloronitrobenzene catabolism pathway in Pseudomonas stutzeri ZWLR2-1

Pseudomonas stutzeri ZWLR2-1 utilizes 2-chloronitrobenzene (2CNB) as a sole source of carbon, nitrogen, and energy. To identify genes involved in this pathway, a 16.2-kb DNA fragment containing putative 2CNB dioxygenase genes was cloned and sequenced. Of the products from the 19 open reading frames that resulted from this fragment, CnbAc and CnbAd exhibited striking identities to the respective α and β subunits of the Nag-like ring-hydroxylating dioxygenases involved in the metabolism of nitrotoluene, nitrobenzene, and naphthalene. The encoding genes were also flanked by two copies of insertion sequence IS6100. CnbAa and CnbAb are similar to the ferredoxin reductase and ferredoxin for anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1. Escherichia coli cells expressing cnbAaAbAcAd converted 2CNB to 3-chlorocatechol with concomitant nitrite release. Cell extracts of E. coli/pCNBC exhibited chlorocatechol 1,2-dioxygenase activity. The cnbCDEF gene cluster, homologous to a 3-chlorocatechol degradation cluster in Sphingomonas sp. strain TFD44, probably contains all of the genes necessary for the conversion of 3-chlorocatechol to 3-oxoadipate. The patchwork-like structure of this catabolic cluster suggests that the cnb cluster for 2CNB degradation evolved by recruiting two catabolic clusters encoding a nitroarene dioxygenase and a chlorocatechol degradation pathway. This provides another example to help elucidate the bacterial evolution of catabolic pathways in response to xenobiotic chemicals.     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway.

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

Chronic contractile activity upregulates the proteasome system in rabbit skeletal muscle.

Remodeling of skeletal muscle in response to altered patterns of contractile activity is achieved, in part, by the regulated degradation of cellular proteins. The ubiquitin-proteasome system is a dominant pathway for protein degradation in eukaryotic cells. To test the role of this pathway in contraction-induced remodeling of skeletal muscle, we used a well-established model of continuous motor nerve stimulation to activate tibialis anterior (TA) muscles of New Zealand White rabbits for periods up to 28 days. Western blot analysis revealed marked and coordinated increases in protein levels of the 20S proteasome and two of its regulatory proteins, PA700 and PA28. mRNA of a representative proteasome subunit also increased coordinately in contracting muscles. Chronic contractile activity of TA also increased total proteasome activity in extracts, as measured by the hydrolysis of a proteasome-specific peptide substrate, and the total capacity of the ubiquitin-proteasome pathway, as measured by the ATP-dependent hydrolysis of an exogenous protein substrate. These results support the potential role of the ubiquitin-proteasome pathway of protein degradation in the contraction-induced remodeling of skeletal muscle.     

Quantitative cell-based protein degradation assays to identify and classify drugs that target the ubiquitin-proteasome system

We have generated a set of dual-reporter human cell lines and devised a chase protocol to quantify proteasomal degradation of a ubiquitin fusion degradation (UFD) substrate, a ubiquitin ligase CRL2(VHL) substrate, and a ubiquitin-independent substrate. Well characterized inhibitors that target different aspects of the ubiquitin-proteasome system can be distinguished by their distinctive patterns of substrate stabilization, enabling assignment of test compounds as inhibitors of the proteasome, ubiquitin chain formation or perception, CRL activity, or the UFD-p97 pathway. We confirmed that degradation of the UFD but not the CRL2(VHL) or ubiquitin-independent substrates depends on p97 activity. We optimized our suite of assays to establish conditions suitable for high-throughput screening and then validated their performance by screening against 160 cell-permeable protein kinase inhibitors. This screen identified Syk inhibitor III as an irreversible p97/vasolin containing protein inhibitor (IC(50) = 1.7 μM) that acts through Cys-522 within the D2 ATPase domain. Our work establishes a high-throughput screening-compatible pipeline for identification and classification of small molecules, cDNAs, or siRNAs that target components of the ubiquitin-proteasome system.     

Calcineurin B subunit interacts with proteasome subunit alpha type 7 and represses hypoxia-inducible factor-1α activity via the proteasome pathway

The calcineurin (CN) B subunit (CNB) is the regulatory subunit of CN, which is the only serine/threonine-specific protein phosphatase regulated by Ca²⁺/CaM. It has been shown to have potential as an anticancer agent, and has a positive effect on the phagocytic index and coefficient. We report here that CNB binds to proteasome subunit alpha type 7 (PSMA7) and inhibits the transactivation activity of hypoxia-inducible factor-1α (HIF-1α) via the proteasome pathway. In addition, we show that CNB represses the expression of vascular endothelial growth factor (VEGF), which is regulated by HIF-1α. These results indicate that CNB modulates cellular proteasome activity via a specific interaction with PSMA7. This may provide a molecular basis for its anticancer and antiviral activities.