Cloning, Expression, and Nucleotide Sequence of thePseudomonas aeruginosa 142 ohb Genes Coding for Oxygenolytic ortho Dehalogenation of Halobenzoates
Cloning, Expression, and Nucleotide Sequence of thePseudomonas aeruginosa 142 ohb Genes Coding for Oxygenolytic ortho Dehalogenation of Halobenzoates
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DOI：
10.1128/aem.65.5.2151-2162.1999
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发表时间：
1999-05
影响因子：
4.4
通讯作者：
T. Tsoi;E. G. Plotnikova;J. Cole;W. Guerin;M. Bagdasarian;J. Tiedje
中科院分区：
文献类型：
--
作者：
T. Tsoi;E. G. Plotnikova;J. Cole;W. Guerin;M. Bagdasarian;J. Tiedje
摘要我们从2-氯苯甲酸（2-CBA）和2，4-二氯苯甲酸（2，4-dCBA）降解菌铜绿假单胞菌142中克隆并鉴定了新的氧解邻位脱卤（ohb）基因。在3，700个大肠杆菌重组子中，有两个克隆DH 5 αF′（pOD 22）和DH 5 αF′（pOD 33）能将2-CBA转化为邻苯二酚，2，4-dCBA和2，5-dCBA转化为4-氯邻苯二酚。含有3，687-bp最小化的ohb DNA区域的pOD 33的亚克隆（质粒pE 43）赋予恶臭假单胞菌PB 2440以2-CBA作为唯一碳源生长的能力。菌株PB 2440（pE 43）也氧化，但在2，4-dCBA、2，5-dCBA或2，6-dCBA上不生长。鉴定了末端氧化还原酶ISPOHB结构基因ohbA和ohbB，它们分别编码分子量为20，253 Da（β-ISP）和48，243 Da（α-ISP）的多肽;这些蛋白质与E.大肠杆菌和铜绿假单胞菌亲本菌株142。邻卤代苯甲酸1，2-双加氧酶活性表现在铁氧还蛋白和还原酶基因的情况下，这表明ISPOHB利用异源宿主提供的电子传递组分。ISPOHB形成了一个新的系统发育簇，其中包括具有非典型结构-功能组织的芳香族加氧酶，并且与主要芳香族加氧酶家族的其他成员相距甚远。一个假定的IclR型调控基因（ohbR）位于ohbAB基因的上游。发现一个未知功能的开放阅读框（ohbC），它与ohbB在长度方向上重叠，但转录方向相反，编码48，969-Da多肽，与大肠杆菌中检测到的49-kDa蛋白质雅阁。杆菌ohb基因的侧翼是IS 1396样序列，其含有在位置4731至5747处的39，715-Da转座酶A（tnpA）的推定基因和在位置346至1563处的45，247-Da DNA拓扑异构酶I/III（上图）的推定基因。Theohb DNA区域在位置56至69和5984至5997处以14-bp不完全反向重复序列为边界。
ABSTRACT We have cloned and characterized novel oxygenolyticortho-dehalogenation (ohb) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degradingPseudomonas aeruginosa 142. Among 3,700 Escherichia coli recombinants, two clones, DH5αF′(pOD22) and DH5αF′(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISPOHBstructural genes ohbA and ohbB, which encode polypeptides with molecular masses of 20,253 Da (β-ISP) and 48,243 Da (α-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized inE. coli and P. aeruginosa parental strain 142. The ortho-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISPOHB utilized electron transfer components provided by the heterologous hosts. ISPOHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (ohbR) was located upstream of the ohbAB genes. An open reading frame (ohbC) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found. TheohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli. The ohbgenes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563. Theohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.