邻苯二甲酸酯降解菌CQ0110Y降解相关基因和蛋白研究
摘要
我室前期从长江和嘉陵江水中检出178种有机污染物,其中邻苯二甲酸酯( Phthalic Acid Esters, PAEs)是主要污染物,浓度达到22.8μg/L;同时,PAEs也是重庆市主城区人群体内的主要污染物,浓度达到78.29μg/L。我们针对PAEs的生物降解开展了相关研究。从污染环境中分离了多株PAEs降解菌,均能以PAEs为唯一碳源和能源进行生长,其中降解菌CQ0110Y的降解效率最高。本实验围绕降解菌CQ0110Y的基本性状、降解相关基因和降解相关蛋白三部分内容展开研究,为CQ0110Y的环境污染治理应用打下了一定基础。
一、邻苯二甲酸酯降解菌CQ0110Y基本性状
1.根据伯杰氏细菌分类手册第八版和第九版中描述的模式菌株的形态学、生理生化特征,结合16S rRNA在genbank中的比对结果,菌株CQ0110Y属于微杆菌属(Microbacterium sp.)。这是国际上首次报道的具有PAEs降解性能的微杆菌属细菌。
2. CQ0110Y进行生物降解反应的最佳pH为6.0～8.0;最佳温度为20～35℃。
3.当DEHP初始浓度低于1350 mg/L时,CQ0110Y对不同初始浓度DEHP的降解反应符合同一指数动力学模型,动力学方程:㏑ C=-0.4087t+A;半减期t1/2= 1.59 d。
4. CQ0110Y对DEHP的降解性能优于其它几种PAEs,对其它PAEs的降解性能随着PAEs側链碳原子的增加而下降。
5.推测DEHP在CQ0110Y菌作用下可能的生物降解途径为:邻苯二甲酸酯在酯酶作用水解成单酯,继续水解成邻苯二甲酸,进一步降解成苯甲酸,苯甲酸在加氧酶作用下形成邻羟基苯甲酸,邻羟基苯甲酸脱羧基生成苯酚,进一步生成邻苯二酚,在1, 2-双加氧酶作用下生成己二烯二酸,进一步生成丙酮酸、琥珀酸等进入三羧酸循环,最终转化为二氧化碳和水。
二、邻苯二甲酸酯降解菌CQ0110Y降解相关基因研究
1.通过限制性内切酶SAU3A I随机酶切细菌基因组DNA,回收3-10 kb范围的DNA片段,采用stratagene公司的基因组文库构建试剂盒(载体为ZAP Express Vector和pBK-CMV Vector,宿主菌是XL1-Blue MRF′strain和XLOLR strain,噬菌体包装试剂为Gigapack III Gold packaging extract),构建了降解菌CQ0110Y的基因组文库。经效价测定,该文库效价达到5 x 107 PFU/μg连接DNA,文库质量高。
2.采用邻苯二甲酸酯琼脂平板特异性选择筛选法获得150个阳性克隆,阳性克隆获得率为1.2x10-4。经过辅助噬菌体作用的单克隆切除,卡那霉素平板及PAEs无机盐培养基双重筛选,最终获得一株阳性克隆菌株。
3.根据载体已知序列设计PCR引物,以重组体质粒为模板,对目的DNA进行PCR扩增,PCR产物纯化后测序,将DNA序列在GenBank中进行登记,取得登记号为EF449771;采用ORF Finder对目的DNA进行分析发现,目的DNA中含有2895 bp的ORF,命名为phtY;经过在Genbank数据库的blast检索比对,phtY与phosphoenolpyruvate Carboxylase(磷酸烯醇丙酮酸脱羧酶)的编码基因高度同源,同源性达到99%。
4.根据目的DNA已知序列设计PCR引物,以CQ0110Y基因组为模板进行PCR扩增,得到1700 bp的DNA片段,与引物设计的DNA大小一致,验证了本实验获得的phtY基因来源菌株CQ0110Y;同时,提取CQ0110Y总RNA,经逆转录成cDNA后进行RT-PCR,也得到1700 bp的DNA片段,说明phtY基因不但来源于菌株CQ0110Y并且能够在细菌内转录成mRNA,可以编码相应的功能蛋白。
5.根据报道的PAEs降解菌DBF63的已知降解相关基因phtC设计相应的引物,以本实验的PAEs降解菌CQ0110Y的基因组为模板,通过PCR扩增相应的功能基因。获得700 bp的DNA片段,经与T载体连接后测序,序列分析显示含有450 bp的ORF,blast检索比对显示为一功能未知的基因,但与phtC基因无同源性,原因不清楚。
三、邻苯二甲酸酯降解菌CQ0110Y降解相关蛋白研究
1.制备CQ0110Y菌分别在LB培养基和PAEs无机盐培养基内生长的菌体总蛋白,分别代表CQ0110Y在PAEs诱导前后的总蛋白。采用二维凝胶电泳分离总蛋白,通过软件分析,诱导前总蛋点有846个,诱导后总蛋白点有847个,诱导后新增加7个蛋白点,消失6个蛋白点,3倍以上表达上调有11个蛋白点,3倍以上表达下调有9个蛋白点,2倍到3倍表达上调有33个蛋白点,2倍到3倍表达下调有38个蛋白点,总共有2倍以上表达差异的蛋白点104个。
2.分别对诱导后2倍以上表达差异的104个蛋白点进行了质谱分析,经过MSCOT搜索引擎检索对差异表达蛋白质进行了鉴定。根据这些功能蛋白的表达情况分析,PAEs降解菌CQ0110Y菌处在PAEs无机盐培养基这个比较特殊的环境中,主要发生了四方面的改变。
(1)为应对PAEs类物质的细胞毒性,细菌增强了对外环境的抗应激及修复能力,增加了触发因子和应急蛋白ClpB等分子伴侣、核糖体蛋白、转肽酶以及核甙酸基转移酶的表达量。
(2)为应对所处的贫营养环境,增强从外界摄取物质及能量的能力,增加了甲酰四氢叶酸酯合成酶、带7蛋白、多糖转运外膜蛋白及外膜孔道蛋白OmpF的表达量。
(3)增强了利用培养基内作为唯一碳源和能源的PAEs的能力,水解酶、加氧酶、脱羧酶等降解功能相关的蛋白酶的表达量由此得到增加。
(4)与LB培养基相比,细菌在PAEs无机盐培养基内生长变缓,许多与生长代谢相关的蛋白酶的表达下调,包括DNA解螺旋酶、RNA聚合酶、DNA错配修复蛋白、组氨酸激酶、天冬氨酸氨甲酰转移酶、UDP-N-乙酰葡糖胺焦磷酸化酶等。
3.本研究结果提示很有必要对这些功能蛋白酶(如MEHP水解酶、加氧酶、双加氧酶、脱羧酶等)继续进行深入研究。
总之,本研究基于从污染环境分离的PAEs降解菌CQ0110Y进行了系列研究,该菌能够利用PAEs作为唯一碳源和能源进行生长,具有高效降解性能,初步阐明了生物降解机理。通过构建并筛选基因组文库获得了PAEs降解相关基因phtY;通过蛋白质谱差异表达蛋白观察到了PAEs降解相关蛋白(MEHP水解酶、加氧酶、双加氧酶、脱羧酶等)并分析了降解菌CQ0110Y在PAEs无机盐培养基内发生的系列变化。该研究与国内外其它相关报道比较,发现降解菌为应对贫营养环境而发生了系列变化,这是以前未见报道的。今后应进一步开展MEHP水解酶、加氧酶、双加氧酶、脱羧酶等功能蛋白的深入研究,以用于PAEs的环境污染治理。
In previous studies we analyzed organic chemicals in water samples from the Yangtze and Jialing Rivers. More than 178 different organic pollutants were detected in these samples; in particular, phthalic acid esters (PAEs) were the main pollutants at a concentration of 22.8μg/l. PAEs were also the main pollutants in a Chongqing population at a concentration of 78.29μg/l in blood. We have also studied the biodegradation of PAEs. Some PAE-degrading bacteria strains were isolated from polluted environments. These strains were able to grow by utilizing PAEs as their sole carbon and energy sources. In particular, strain CQ0110Y exhibited the highest PAE degradation efficiency. In the present study, we investigated the basic characteristics of CQ0110Y and examined one gene and many proteins associated with PAE degradation.
Part I. Study of the basic characteristics of CQ0110Y
1. Morphological analysis, physico-biochemical characterization and genetic specificity tests revealed that strain CQ0110Y was a Microbacterium sp. This is the first reported case of DEHP degradation by Microbacterium sp. strain.
2. The optimum conditions for CQ0110Y degradation of DEHP were pH 6.0–8.0 and a temperature of 20–35°C.
3. DEHP degradation can be described by a single exponential model for an initial DEHP concentration of <1350 mg/l. The kinetics equation was lnC=?0.4087t+A, with a degradation half-life of DEHP in wastewater of 1.59 days.
4. Degradation by CQ0110Y was more efficient for DEHP than for other PAEs. PAE degradation becomes more difficult with increasing carbon chain length.
5. A degradation pathway for DEHP in strain CQ0110Y is proposed as follows: DEHP is hydrolyzed to monoethylhexylphthalate and phthalic acid by the action of esterase. Benzenecarboxylic acid is then formed via the decarboxylation of phthalic acid. Hydroxylation at consecutive positions in benzenecarboxylic acid yields orthohydroxybenzoic acid, followed by pyrocatechin and muconic acid. After the tricarboxylic acid cycle, the terminal degradation products are CO2 and H2O.
Part II. Study of a degradation-associated gene in CQ0110Y
1. A genomic library of the CQ0110Y strain was constructed using DNA fragments of 3–10 kb. The DNA fragments were randomly excised from the bacterial genome using the restriction enzyme SAU3A I. A ZAP Express Predigested Vector Kit and ZAP Express Predigested Gigapack Clone Kit were used for genomic library construction. The high titer of the genomic library (5×10~7 PFU/μg ligated DNA) confirmed the high quality of the genomic library.
2. There were 150 positive clones screened from the genomic library on PAE agar plates, with a pick-up rate for positive clones of 1.2×10–4. The positive clones were used for single-clone excision and were doubly screened on agar plates containing kanamycin and PAE inorganic salt culture. A positive clone was finally obtained.
3. PCR primers were designed from the known vector sequence and a recombinant plasmid was used as the template for PCR amplification of the target DNA, which was recovered from the PCR product and used for sequencing. The sequence was registered in GenBank under accession number EF449771. There was an open reading frame of 2895 bp in the sequence according to ORF Finder analysis. We designated the gene as phtY. Blast results demonstrated that the phtY gene has 99% homology to the gene coding for phosphoenolpyruvate carboxylase.
4. PCR primers were designed from the known sequence of the phtY gene and the CQ0110Y genome was used as the template for PCR amplification. The size of the PCR product was 1700 bp, in agreement with the size designed. The results demonstrated that the phtY gene originated from the CQ0110Y strain. Total RNA was also extracted from CQ0110Y and used for reverse transcription to obtain the corresponding cDNA. RT-PCR yielded a product of 1700 bp. The results demonstrated that the phtY gene not only originates from CQ0110Y, but can also be transcribed to mRNA and code for a functional protein.
5. PCR primers were designed from the known sequence of the phtC gene from another PAE-degrading bacterium (DBF63) and the CQ0110Y genome was used as the template for PCR amplification. The size of the PCR product was 700 bp. There was an open reading frame of 450 bp in the sequence according to ORF Finder analysis. Blast results demonstrated that this gene codes for a protein of unknown function, but has no homology to the phtC gene of DBF63.
Part III. Study of degradation-associated proteins in CQ0110Y
1. Total proteins were harvested from CQ0110Y cultivated in LB culture and PAE inorganic salt culture, representing total proteins without and with induction by PAEs, respectively. Two-dimensional electrophoresis (2-DE) was used to separate the proteins. ImageMaster 2D Platinum 5.0 analysis revealed 846 different proteins in non-treated CQ0110Y and 843 in PAE-treated CQ0110Y. Seven new proteins appeared and six proteins disappeared after PAE treatment. The expression of 11 proteins was up-regulated by a factor of more than three and that of nine proteins was down-regulated by more than a factor of three after PAE treatment. A further 33 proteins exhibited expression up-regulation of two- to three-fold and 38 proteins exhibited expression down-regulation by a similar factor after PAE treatment. Finally, the expression of 104 proteins exhibited more than two-fold up- or down-regulation on PAE induction.
2. The 104 proteins expressed more than two-fold up- or down-regulation on PAE induction were identified by mass spectrometric analysis. According to the up- or down-regulation of those functional proteins, a conclusion could be made that there were four major changes of CQ0110Y strain cultivated in PAEs inorganic salt culture in comparison with LB culture.
(1) CQ0110Y strain had enhanced the ability of repairing itself and resisting to stress press to cope with the damage caused by PAEs. The expression of Trigger factor、clpB protein、ribosomal protein、transpeptidase and nucleotidyltransferase was up-regulation, respectively.
(2) CQ0110Y strain had enhanced the ability of uptaking substances and energy from surrounding to cope with the deficient environment. The expression of Formyltetra -hydrofolate synthetase、band 7 protein、outer membrane protein and ompF Porin was up-regulation, respectively.
(3) The PAEs degradation-associated proteins of CQ0110Y strain were up-regulation expression. Those proteins included monoethylhexylphthalate hydrolase (MEHP hydrolase), oxygenase, dioxygenase, decarboxylase and so on.
(4) The slow growth of CQ0110Y strain in PAEs inorganic salt culture had made the down-regulation expression of the metabolism--associated proteins, such as DNA helicase、UvrD/REP helicase、RNA polymerase、DNA mismatch repair protein、Histidine kinase、 Aspartate carbamoyltransferase and UDP-N-acetylglucosamine pyrophosphorylase.
3. The results indicate that a detailed investigation of these functional enzymes(MEHP hydrolase、oxygenase、dioxygenase、decarboxylase) is essential.
In a word, a PAE-degrading bacteria strains was isolated from polluted environment. The strain, which exhibited a high PAE degradation efficiency, was able to grow by utilizing PAEs as their sole carbon and energy sources. The supposed degradation pathway of PAEs by strain CQ0110Y was described. A degradation-associated gene in CQ0110Y, designated as phtY, was obtained from the genomic library of the CQ0110Y strain. Some degradation-associated proteins in CQ0110Y, such as MEHP hydrolase、oxygenase、dioxygenase、decarboxylase, were identified by mass spectrometric analysis. A detailed investigation of these functional enzymes is essential.
一、邻苯二甲酸酯降解菌CQ0110Y基本性状
1.根据伯杰氏细菌分类手册第八版和第九版中描述的模式菌株的形态学、生理生化特征,结合16S rRNA在genbank中的比对结果,菌株CQ0110Y属于微杆菌属(Microbacterium sp.)。这是国际上首次报道的具有PAEs降解性能的微杆菌属细菌。
2. CQ0110Y进行生物降解反应的最佳pH为6.0～8.0;最佳温度为20～35℃。
3.当DEHP初始浓度低于1350 mg/L时,CQ0110Y对不同初始浓度DEHP的降解反应符合同一指数动力学模型,动力学方程:㏑ C=-0.4087t+A;半减期t1/2= 1.59 d。
4. CQ0110Y对DEHP的降解性能优于其它几种PAEs,对其它PAEs的降解性能随着PAEs側链碳原子的增加而下降。
5.推测DEHP在CQ0110Y菌作用下可能的生物降解途径为:邻苯二甲酸酯在酯酶作用水解成单酯,继续水解成邻苯二甲酸,进一步降解成苯甲酸,苯甲酸在加氧酶作用下形成邻羟基苯甲酸,邻羟基苯甲酸脱羧基生成苯酚,进一步生成邻苯二酚,在1, 2-双加氧酶作用下生成己二烯二酸,进一步生成丙酮酸、琥珀酸等进入三羧酸循环,最终转化为二氧化碳和水。
二、邻苯二甲酸酯降解菌CQ0110Y降解相关基因研究
1.通过限制性内切酶SAU3A I随机酶切细菌基因组DNA,回收3-10 kb范围的DNA片段,采用stratagene公司的基因组文库构建试剂盒(载体为ZAP Express Vector和pBK-CMV Vector,宿主菌是XL1-Blue MRF′strain和XLOLR strain,噬菌体包装试剂为Gigapack III Gold packaging extract),构建了降解菌CQ0110Y的基因组文库。经效价测定,该文库效价达到5 x 107 PFU/μg连接DNA,文库质量高。
2.采用邻苯二甲酸酯琼脂平板特异性选择筛选法获得150个阳性克隆,阳性克隆获得率为1.2x10-4。经过辅助噬菌体作用的单克隆切除,卡那霉素平板及PAEs无机盐培养基双重筛选,最终获得一株阳性克隆菌株。
3.根据载体已知序列设计PCR引物,以重组体质粒为模板,对目的DNA进行PCR扩增,PCR产物纯化后测序,将DNA序列在GenBank中进行登记,取得登记号为EF449771;采用ORF Finder对目的DNA进行分析发现,目的DNA中含有2895 bp的ORF,命名为phtY;经过在Genbank数据库的blast检索比对,phtY与phosphoenolpyruvate Carboxylase(磷酸烯醇丙酮酸脱羧酶)的编码基因高度同源,同源性达到99%。
4.根据目的DNA已知序列设计PCR引物,以CQ0110Y基因组为模板进行PCR扩增,得到1700 bp的DNA片段,与引物设计的DNA大小一致,验证了本实验获得的phtY基因来源菌株CQ0110Y;同时,提取CQ0110Y总RNA,经逆转录成cDNA后进行RT-PCR,也得到1700 bp的DNA片段,说明phtY基因不但来源于菌株CQ0110Y并且能够在细菌内转录成mRNA,可以编码相应的功能蛋白。
5.根据报道的PAEs降解菌DBF63的已知降解相关基因phtC设计相应的引物,以本实验的PAEs降解菌CQ0110Y的基因组为模板,通过PCR扩增相应的功能基因。获得700 bp的DNA片段,经与T载体连接后测序,序列分析显示含有450 bp的ORF,blast检索比对显示为一功能未知的基因,但与phtC基因无同源性,原因不清楚。
三、邻苯二甲酸酯降解菌CQ0110Y降解相关蛋白研究
1.制备CQ0110Y菌分别在LB培养基和PAEs无机盐培养基内生长的菌体总蛋白,分别代表CQ0110Y在PAEs诱导前后的总蛋白。采用二维凝胶电泳分离总蛋白,通过软件分析,诱导前总蛋点有846个,诱导后总蛋白点有847个,诱导后新增加7个蛋白点,消失6个蛋白点,3倍以上表达上调有11个蛋白点,3倍以上表达下调有9个蛋白点,2倍到3倍表达上调有33个蛋白点,2倍到3倍表达下调有38个蛋白点,总共有2倍以上表达差异的蛋白点104个。
2.分别对诱导后2倍以上表达差异的104个蛋白点进行了质谱分析,经过MSCOT搜索引擎检索对差异表达蛋白质进行了鉴定。根据这些功能蛋白的表达情况分析,PAEs降解菌CQ0110Y菌处在PAEs无机盐培养基这个比较特殊的环境中,主要发生了四方面的改变。
(1)为应对PAEs类物质的细胞毒性,细菌增强了对外环境的抗应激及修复能力,增加了触发因子和应急蛋白ClpB等分子伴侣、核糖体蛋白、转肽酶以及核甙酸基转移酶的表达量。
(2)为应对所处的贫营养环境,增强从外界摄取物质及能量的能力,增加了甲酰四氢叶酸酯合成酶、带7蛋白、多糖转运外膜蛋白及外膜孔道蛋白OmpF的表达量。
(3)增强了利用培养基内作为唯一碳源和能源的PAEs的能力,水解酶、加氧酶、脱羧酶等降解功能相关的蛋白酶的表达量由此得到增加。
(4)与LB培养基相比,细菌在PAEs无机盐培养基内生长变缓,许多与生长代谢相关的蛋白酶的表达下调,包括DNA解螺旋酶、RNA聚合酶、DNA错配修复蛋白、组氨酸激酶、天冬氨酸氨甲酰转移酶、UDP-N-乙酰葡糖胺焦磷酸化酶等。
3.本研究结果提示很有必要对这些功能蛋白酶(如MEHP水解酶、加氧酶、双加氧酶、脱羧酶等)继续进行深入研究。
总之,本研究基于从污染环境分离的PAEs降解菌CQ0110Y进行了系列研究,该菌能够利用PAEs作为唯一碳源和能源进行生长,具有高效降解性能,初步阐明了生物降解机理。通过构建并筛选基因组文库获得了PAEs降解相关基因phtY;通过蛋白质谱差异表达蛋白观察到了PAEs降解相关蛋白(MEHP水解酶、加氧酶、双加氧酶、脱羧酶等)并分析了降解菌CQ0110Y在PAEs无机盐培养基内发生的系列变化。该研究与国内外其它相关报道比较,发现降解菌为应对贫营养环境而发生了系列变化,这是以前未见报道的。今后应进一步开展MEHP水解酶、加氧酶、双加氧酶、脱羧酶等功能蛋白的深入研究,以用于PAEs的环境污染治理。
In previous studies we analyzed organic chemicals in water samples from the Yangtze and Jialing Rivers. More than 178 different organic pollutants were detected in these samples; in particular, phthalic acid esters (PAEs) were the main pollutants at a concentration of 22.8μg/l. PAEs were also the main pollutants in a Chongqing population at a concentration of 78.29μg/l in blood. We have also studied the biodegradation of PAEs. Some PAE-degrading bacteria strains were isolated from polluted environments. These strains were able to grow by utilizing PAEs as their sole carbon and energy sources. In particular, strain CQ0110Y exhibited the highest PAE degradation efficiency. In the present study, we investigated the basic characteristics of CQ0110Y and examined one gene and many proteins associated with PAE degradation.
Part I. Study of the basic characteristics of CQ0110Y
1. Morphological analysis, physico-biochemical characterization and genetic specificity tests revealed that strain CQ0110Y was a Microbacterium sp. This is the first reported case of DEHP degradation by Microbacterium sp. strain.
2. The optimum conditions for CQ0110Y degradation of DEHP were pH 6.0–8.0 and a temperature of 20–35°C.
3. DEHP degradation can be described by a single exponential model for an initial DEHP concentration of <1350 mg/l. The kinetics equation was lnC=?0.4087t+A, with a degradation half-life of DEHP in wastewater of 1.59 days.
4. Degradation by CQ0110Y was more efficient for DEHP than for other PAEs. PAE degradation becomes more difficult with increasing carbon chain length.
5. A degradation pathway for DEHP in strain CQ0110Y is proposed as follows: DEHP is hydrolyzed to monoethylhexylphthalate and phthalic acid by the action of esterase. Benzenecarboxylic acid is then formed via the decarboxylation of phthalic acid. Hydroxylation at consecutive positions in benzenecarboxylic acid yields orthohydroxybenzoic acid, followed by pyrocatechin and muconic acid. After the tricarboxylic acid cycle, the terminal degradation products are CO2 and H2O.
Part II. Study of a degradation-associated gene in CQ0110Y
1. A genomic library of the CQ0110Y strain was constructed using DNA fragments of 3–10 kb. The DNA fragments were randomly excised from the bacterial genome using the restriction enzyme SAU3A I. A ZAP Express Predigested Vector Kit and ZAP Express Predigested Gigapack Clone Kit were used for genomic library construction. The high titer of the genomic library (5×10~7 PFU/μg ligated DNA) confirmed the high quality of the genomic library.
2. There were 150 positive clones screened from the genomic library on PAE agar plates, with a pick-up rate for positive clones of 1.2×10–4. The positive clones were used for single-clone excision and were doubly screened on agar plates containing kanamycin and PAE inorganic salt culture. A positive clone was finally obtained.
3. PCR primers were designed from the known vector sequence and a recombinant plasmid was used as the template for PCR amplification of the target DNA, which was recovered from the PCR product and used for sequencing. The sequence was registered in GenBank under accession number EF449771. There was an open reading frame of 2895 bp in the sequence according to ORF Finder analysis. We designated the gene as phtY. Blast results demonstrated that the phtY gene has 99% homology to the gene coding for phosphoenolpyruvate carboxylase.
4. PCR primers were designed from the known sequence of the phtY gene and the CQ0110Y genome was used as the template for PCR amplification. The size of the PCR product was 1700 bp, in agreement with the size designed. The results demonstrated that the phtY gene originated from the CQ0110Y strain. Total RNA was also extracted from CQ0110Y and used for reverse transcription to obtain the corresponding cDNA. RT-PCR yielded a product of 1700 bp. The results demonstrated that the phtY gene not only originates from CQ0110Y, but can also be transcribed to mRNA and code for a functional protein.
5. PCR primers were designed from the known sequence of the phtC gene from another PAE-degrading bacterium (DBF63) and the CQ0110Y genome was used as the template for PCR amplification. The size of the PCR product was 700 bp. There was an open reading frame of 450 bp in the sequence according to ORF Finder analysis. Blast results demonstrated that this gene codes for a protein of unknown function, but has no homology to the phtC gene of DBF63.
Part III. Study of degradation-associated proteins in CQ0110Y
1. Total proteins were harvested from CQ0110Y cultivated in LB culture and PAE inorganic salt culture, representing total proteins without and with induction by PAEs, respectively. Two-dimensional electrophoresis (2-DE) was used to separate the proteins. ImageMaster 2D Platinum 5.0 analysis revealed 846 different proteins in non-treated CQ0110Y and 843 in PAE-treated CQ0110Y. Seven new proteins appeared and six proteins disappeared after PAE treatment. The expression of 11 proteins was up-regulated by a factor of more than three and that of nine proteins was down-regulated by more than a factor of three after PAE treatment. A further 33 proteins exhibited expression up-regulation of two- to three-fold and 38 proteins exhibited expression down-regulation by a similar factor after PAE treatment. Finally, the expression of 104 proteins exhibited more than two-fold up- or down-regulation on PAE induction.
2. The 104 proteins expressed more than two-fold up- or down-regulation on PAE induction were identified by mass spectrometric analysis. According to the up- or down-regulation of those functional proteins, a conclusion could be made that there were four major changes of CQ0110Y strain cultivated in PAEs inorganic salt culture in comparison with LB culture.
(1) CQ0110Y strain had enhanced the ability of repairing itself and resisting to stress press to cope with the damage caused by PAEs. The expression of Trigger factor、clpB protein、ribosomal protein、transpeptidase and nucleotidyltransferase was up-regulation, respectively.
(2) CQ0110Y strain had enhanced the ability of uptaking substances and energy from surrounding to cope with the deficient environment. The expression of Formyltetra -hydrofolate synthetase、band 7 protein、outer membrane protein and ompF Porin was up-regulation, respectively.
(3) The PAEs degradation-associated proteins of CQ0110Y strain were up-regulation expression. Those proteins included monoethylhexylphthalate hydrolase (MEHP hydrolase), oxygenase, dioxygenase, decarboxylase and so on.
(4) The slow growth of CQ0110Y strain in PAEs inorganic salt culture had made the down-regulation expression of the metabolism--associated proteins, such as DNA helicase、UvrD/REP helicase、RNA polymerase、DNA mismatch repair protein、Histidine kinase、 Aspartate carbamoyltransferase and UDP-N-acetylglucosamine pyrophosphorylase.
3. The results indicate that a detailed investigation of these functional enzymes(MEHP hydrolase、oxygenase、dioxygenase、decarboxylase) is essential.
In a word, a PAE-degrading bacteria strains was isolated from polluted environment. The strain, which exhibited a high PAE degradation efficiency, was able to grow by utilizing PAEs as their sole carbon and energy sources. The supposed degradation pathway of PAEs by strain CQ0110Y was described. A degradation-associated gene in CQ0110Y, designated as phtY, was obtained from the genomic library of the CQ0110Y strain. Some degradation-associated proteins in CQ0110Y, such as MEHP hydrolase、oxygenase、dioxygenase、decarboxylase, were identified by mass spectrometric analysis. A detailed investigation of these functional enzymes is essential.
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