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Characterization of arsenic resistance genes in Rhodococcus jostii RHA1
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
A response regulator BphQ, whose orthologs are present in related beta-proteobacteria, has been found to play important role in catabolite control in KKS102. In ATCC 17616 pE promoter activity was also under control of BphQ. These findings suggested that ICE-specified pE promoter is able to respond to different substrates depending on the host in which it resides. We also present evidences that metabolic conversions of glucose or galactose are crucial to pE repression in ATCC 17616. doi:10.1016/j.jbiotec.2010.09.099 [P-E.78] Isolation of novel (+)-catechin degrading bacterium, Burkholderia oxyphila sp. nov. OX-O1, from acidic forest soil and its (+)-catechin metabolic pathway Y Otsuka 1,∗ , H Murata 1 , M Matsuda 2 , T Sonoki 3 , M Nakamura 1 1
Forestry and Forest Products Research Institute, Japan Toyama Prefectural University, Japan 3 Hirosaki University, Japan Keywords: catechin; Burkholderia; acidic forest soil; biomass 2
Forest soil contains various persistent compounds derived from plant materials. We isolated a novel (+)-catechin degrading bacterium, Burkholderia oxyphila sp. nov. OX-01 (OX-01) as an example of a bacterium capable of degrading persistent aromatic compounds in forest soil. This bacterium was isolated by emichment technique and grew on (+)-catechin as sole carbon source under acidic condition (pH3.5). Crude enzyme reaction and structural study of its product showed that (+)-catechin is biotransformed into taxifolin during the preliminary stages of its metabolism by OX-01. HPLC and GC-MS analysis indicated that a crude enzyme isolated from the OX-01 transformed (+)-catechin to protocatechuic acid via leucocyanidin and taxifolin. In addition, both catechin 4-hydroxylase and leucocyanidin 4-dehydrogenase were localized in cytosol. Moreover, these enzymes require the 2R-flavan 3-ol structure and do not require the conformation of C-3 for C-4 oxidation. In the plant, most flavanol unit of condensed tannin is 2R-stereoisomer, and in relative stereochemistry of C-2 and C-3, both cis and trans isomers exist. Thus, it is considered that the function of catechin carbonylation in OX-01 is evolving to adapt for the plant producing catechins. This is a first report on biotransformation of (+)-catechin into protocatechuic acid via leucocyanidin and taxifolin by aerobic bacterium. This report suggests that acidic forest soil can become unique resource of natural aromatic compound degrading microorganisms. doi:10.1016/j.jbiotec.2010.09.100 [P-E.79] Characterization of arsenic resistance genes in Rhodococcus jostii RHA1 K. Miyauchi 1,∗ , M. Fujita 1 , K. Oikawa 1 , Y. Ohtomo 1 , G. Endo 1 , M. Fukuda 2 1
Department Civil and Environ. Eng., Tohoku Gakuin Univ, Japan Department Bioeng., Nagaoka University Tech., Japan Keywords: arsenic; rhodococcus; gene expression; bioremediation 2
Arsenic is one of metalloids and widely distributed throughout the world. Bacteria have various mechanisms to resist to arsenic, especially to arsenite and arsenate, which are stable arsenical species. Rhodococcus jostii RHA1 is a polychlorinated biphenyl
S241
degrader, and its genome sequence was determined recently. In its genome sequence, three open reading frames (ro03367, ro03368, and ro03369) were found which showed similarities with arsenic resistance genes and they were designated arsR, arsB, and arsC, respectively. In this study, we showed the involvement of these genes in the arsenic resistance of RHA1 and estimated the ArsRbinding site using the reporter assay. To check the resistance of RHA1 toward arsenite or arsenate, RHA1 was incubated in LB medium containing various concentrations of arsenite or arsenate. RHA1 was resistant to 3 mM arsenite and 10 mM arsenate, and its arsRBC - deletion mutant was not resistant to 0.1 mM arsenite and 1 mM arsenate. Introducing DNA fragment containing arsRBC to the mutant complemented the resistance of the mutant to arsenic. The promoter region of arsR was amplified by PCR and inserted upstream of the luciferase gene in a promoter probe vector pKLA1. The luciferase activities of RHA1 transformants harboring the resultant plasmids were measured in the presence of arsenate. It was indicated that the luciferase activity increased in the presence of arsenate. By shortening the promoter region inserted in the promoter probe vector, it was suggested that the ArsR - binding region is located just upstream the start codon of the presumed start codon of arsR. These results showed that the arsRBC genes are responsible for the resistance to arsenic in RHA1. doi:10.1016/j.jbiotec.2010.09.101 [P-E.80] Substrate interactions during the biodegradation of benzene and toluene hydrocarbons at various salinities Ching-Hsing Lin 2,∗ , Chi-Yuan Lee 1 , Wen Der Liu 2 , Yu-Jie Chang 3 1
National Taiwan Ocean University, Taiwan Tungnan University, Taiwan 3 Graduate School of Environmental Education & Resources, Taipei Municipal University of Education, Taiwan Keywords: Biodegradation; Substrate interactions; Salinity; Kinetics 2
The purposes of this research were to investigate the complex effects of the interaction behaviours between substrates at various salinities. Experiment work using the enrichment cultures to degrade the multiple benzene and toluene under a particular salinity ranging from 0-50 g l-1NaCl in batch mode. The obtained microbial kinetic constants of binary substrate under various salinities were modeled using noncompetitive inhibition kinetics. The result shows that toluene become the inhibition substrate for benzene in NaCl solution (0 g l-1). As salinity increases, the inhibition behaviour of substrates becomes less obvious. Especially when salinity increases at 40-50 g l-1 NaCl, toluene will change from inhibition substrate for benzene to promote degradation. In high salinity solution, toluene was promoting obviously the degradation of benzene. The reason was estimated that in high salinity, the growth of the bacteria was strongly inhibited by salinity. When there is certain substrate, like toluene that is easily used for growth by bacteria, it will provide the enzyme that is needed for decomposing benzene. Therefore, in the toluene mixed solution of benzene, the depletion rate will be faster than in sole benzene solution. Salinity becomes the main inhibition for substrate degradation. The benzene and toluene concentration showed in COD unit indicates that the value of salinity inhibition constant on multiple substrate degradation T (S ) K is 26.7 g l-1 and the salinity inhibition constant on bacterial growth T () K is 17.4 g l-1. That the value of T (S ) K is higher than that of T () K shows that the bacterial growth is inhibited more strongly by salinity than substrate degradation.
A response regulator BphQ, whose orthologs are present in related beta-proteobacteria, has been found to play important role in catabolite control in KKS102. In ATCC 17616 pE promoter activity was also under control of BphQ. These findings suggested that ICE-specified pE promoter is able to respond to different substrates depending on the host in which it resides. We also present evidences that metabolic conversions of glucose or galactose are crucial to pE repression in ATCC 17616. doi:10.1016/j.jbiotec.2010.09.099 [P-E.78] Isolation of novel (+)-catechin degrading bacterium, Burkholderia oxyphila sp. nov. OX-O1, from acidic forest soil and its (+)-catechin metabolic pathway Y Otsuka 1,∗ , H Murata 1 , M Matsuda 2 , T Sonoki 3 , M Nakamura 1 1
Forestry and Forest Products Research Institute, Japan Toyama Prefectural University, Japan 3 Hirosaki University, Japan Keywords: catechin; Burkholderia; acidic forest soil; biomass 2
Forest soil contains various persistent compounds derived from plant materials. We isolated a novel (+)-catechin degrading bacterium, Burkholderia oxyphila sp. nov. OX-01 (OX-01) as an example of a bacterium capable of degrading persistent aromatic compounds in forest soil. This bacterium was isolated by emichment technique and grew on (+)-catechin as sole carbon source under acidic condition (pH3.5). Crude enzyme reaction and structural study of its product showed that (+)-catechin is biotransformed into taxifolin during the preliminary stages of its metabolism by OX-01. HPLC and GC-MS analysis indicated that a crude enzyme isolated from the OX-01 transformed (+)-catechin to protocatechuic acid via leucocyanidin and taxifolin. In addition, both catechin 4-hydroxylase and leucocyanidin 4-dehydrogenase were localized in cytosol. Moreover, these enzymes require the 2R-flavan 3-ol structure and do not require the conformation of C-3 for C-4 oxidation. In the plant, most flavanol unit of condensed tannin is 2R-stereoisomer, and in relative stereochemistry of C-2 and C-3, both cis and trans isomers exist. Thus, it is considered that the function of catechin carbonylation in OX-01 is evolving to adapt for the plant producing catechins. This is a first report on biotransformation of (+)-catechin into protocatechuic acid via leucocyanidin and taxifolin by aerobic bacterium. This report suggests that acidic forest soil can become unique resource of natural aromatic compound degrading microorganisms. doi:10.1016/j.jbiotec.2010.09.100 [P-E.79] Characterization of arsenic resistance genes in Rhodococcus jostii RHA1 K. Miyauchi 1,∗ , M. Fujita 1 , K. Oikawa 1 , Y. Ohtomo 1 , G. Endo 1 , M. Fukuda 2 1
Department Civil and Environ. Eng., Tohoku Gakuin Univ, Japan Department Bioeng., Nagaoka University Tech., Japan Keywords: arsenic; rhodococcus; gene expression; bioremediation 2
Arsenic is one of metalloids and widely distributed throughout the world. Bacteria have various mechanisms to resist to arsenic, especially to arsenite and arsenate, which are stable arsenical species. Rhodococcus jostii RHA1 is a polychlorinated biphenyl
S241
degrader, and its genome sequence was determined recently. In its genome sequence, three open reading frames (ro03367, ro03368, and ro03369) were found which showed similarities with arsenic resistance genes and they were designated arsR, arsB, and arsC, respectively. In this study, we showed the involvement of these genes in the arsenic resistance of RHA1 and estimated the ArsRbinding site using the reporter assay. To check the resistance of RHA1 toward arsenite or arsenate, RHA1 was incubated in LB medium containing various concentrations of arsenite or arsenate. RHA1 was resistant to 3 mM arsenite and 10 mM arsenate, and its arsRBC - deletion mutant was not resistant to 0.1 mM arsenite and 1 mM arsenate. Introducing DNA fragment containing arsRBC to the mutant complemented the resistance of the mutant to arsenic. The promoter region of arsR was amplified by PCR and inserted upstream of the luciferase gene in a promoter probe vector pKLA1. The luciferase activities of RHA1 transformants harboring the resultant plasmids were measured in the presence of arsenate. It was indicated that the luciferase activity increased in the presence of arsenate. By shortening the promoter region inserted in the promoter probe vector, it was suggested that the ArsR - binding region is located just upstream the start codon of the presumed start codon of arsR. These results showed that the arsRBC genes are responsible for the resistance to arsenic in RHA1. doi:10.1016/j.jbiotec.2010.09.101 [P-E.80] Substrate interactions during the biodegradation of benzene and toluene hydrocarbons at various salinities Ching-Hsing Lin 2,∗ , Chi-Yuan Lee 1 , Wen Der Liu 2 , Yu-Jie Chang 3 1
National Taiwan Ocean University, Taiwan Tungnan University, Taiwan 3 Graduate School of Environmental Education & Resources, Taipei Municipal University of Education, Taiwan Keywords: Biodegradation; Substrate interactions; Salinity; Kinetics 2
The purposes of this research were to investigate the complex effects of the interaction behaviours between substrates at various salinities. Experiment work using the enrichment cultures to degrade the multiple benzene and toluene under a particular salinity ranging from 0-50 g l-1NaCl in batch mode. The obtained microbial kinetic constants of binary substrate under various salinities were modeled using noncompetitive inhibition kinetics. The result shows that toluene become the inhibition substrate for benzene in NaCl solution (0 g l-1). As salinity increases, the inhibition behaviour of substrates becomes less obvious. Especially when salinity increases at 40-50 g l-1 NaCl, toluene will change from inhibition substrate for benzene to promote degradation. In high salinity solution, toluene was promoting obviously the degradation of benzene. The reason was estimated that in high salinity, the growth of the bacteria was strongly inhibited by salinity. When there is certain substrate, like toluene that is easily used for growth by bacteria, it will provide the enzyme that is needed for decomposing benzene. Therefore, in the toluene mixed solution of benzene, the depletion rate will be faster than in sole benzene solution. Salinity becomes the main inhibition for substrate degradation. The benzene and toluene concentration showed in COD unit indicates that the value of salinity inhibition constant on multiple substrate degradation T (S ) K is 26.7 g l-1 and the salinity inhibition constant on bacterial growth T () K is 17.4 g l-1. That the value of T (S ) K is higher than that of T () K shows that the bacterial growth is inhibited more strongly by salinity than substrate degradation.