Occurrence of Tn4371-Related Mobile Elements and Sequences in (Chloro)biphenyl-Degrading Bacteria

Tn4371, a 55-kb transposable element involved in the degradation and biphenyl or 4-chlorobiphenyl identified in Ralstonia eutropha A5, displays a modular structure including a phage-like integrase gene (int), a Pseudomonas-like (chloro)biphenyl catabolic gene cluster (bph), and RP4- and Ti-plasmid-like transfer genes (trb) (C. Merlin, D. Springael, and A. Toussaint, Plasmid 41:40–54, 1999). Southern blot hybridization was used to examine the presence of different regions of Tn4371 in a collection of (chloro)biphenyl-degrading bacteria originating from different habitats and belonging to different bacterial genera. Tn4371-related sequences were never detected on endogenous plasmids. Although the gene probes containing only bph sequences hybridized to genomic DNA from most strains tested, a limited selection of strains, all β-proteobacteria, displayed hybridization patterns similar to the Tn4371 bph cluster. Homology between Tn4371 and DNA of two of those strains, originating from the same area as strain A5, extended outside the catabolic genes and covered the putative transfer region of Tn4371. On the other hand, none of the (chloro)biphenyl degraders hybridized with the outer left part of Tn4371 containing the int gene. The bph catabolic determinant of the two strains displaying homology to the Tn4371 transfer genes and a third strain isolated from the A5 area could be mobilized to a R. eutropha recipient, after insertion into an endogenous or introduced IncP1 plasmid. The mobilized DNA of those strains included all Tn4371 homologous sequences previously identified in their genome. Our observations show that the bph genes present on Tn4371 are highly conserved between different (chloro)biphenyl-degrading hosts, isolated globally but belonging mainly to the β-proteobacteria. On the other hand, Tn4371-related mobile elements carrying bph genes are apparently only found in isolates from the environment that provided the Tn4371-bearing isolate A5.

Tn4371是一种55 kb的转座因子（transposable element），参与联苯（biphenyl）与4-氯联苯（4-chlorobiphenyl）的降解过程，该因子发现于真养产碱菌（Ralstonia eutropha）A5菌株中，具备模块化结构，包含类噬菌体整合酶基因（int，phage-like integrase gene）、假单胞菌属类（氯）联苯分解代谢基因簇（bph，Pseudomonas-like (chloro)biphenyl catabolic gene cluster）以及RP4与Ti质粒类转移基因（trb，RP4- and Ti-plasmid-like transfer genes）（引自C. Merlin、D. Springael与A. Toussaint发表于《Plasmid》第41卷第40-54页，1999年的研究）。研究采用Southern印迹杂交（Southern blot hybridization）技术，对采集自不同生境、隶属于不同细菌属的一批（氯）联苯降解细菌菌株开展Tn4371不同区域的存在性检测，结果显示Tn4371相关序列从未在内源质粒中被检出。尽管仅包含bph序列的基因探针可与多数受试菌株的基因组DNA发生杂交，但仅有有限的数株β-变形菌门（β-proteobacteria）菌株呈现出与Tn4371的bph基因簇高度相似的杂交图谱。其中两株与A5菌株同环境分离的菌株，其DNA与Tn4371的同源序列不仅覆盖分解代谢基因区域，还延伸至Tn4371的推定转移区域；而另一方面，所有受试的（氯）联苯降解菌株均未与Tn4371左侧外侧携带int基因的区域发生杂交。这两株与Tn4371转移基因具有同源性的菌株，以及另一株从A5菌株所在环境分离的菌株，其bph分解代谢决定簇在插入内源质粒或外源IncP1质粒（IncP1 plasmid）后，可被转移至真养产碱菌受体菌株中，上述菌株的转移DNA包含了先前在其基因组中鉴定出的全部Tn4371同源序列。本研究结果表明，Tn4371上携带的bph基因在不同（氯）联苯降解宿主间具有高度保守性，这些宿主虽在全球范围内分离得到，但主要隶属于β-变形菌门；而携带bph基因的Tn4371相关可移动元件，显然仅存在于分离出携带Tn4371的A5菌株的原环境分离株中。