Study Of A Gene And Proteins Associated With PAEs Degradation In Strain CQ0110Y

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 CQ0110Y1. 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 CQ0110Y1. 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 CQ0110Y1. 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.