嗜高溫Geobacillus屬菌種分子親緣特性研究

Abstract

Geobacillus屬是2001年新命名的一類細菌，具嗜熱、兼性厭氧、降解烴和產生表面活性劑等特性，除在微生物採油、環境治理等領域具有應用潛力外；此類細菌具有特殊的功能基因與酵素，於分子遺傳工程上具有重要的研究價值。由於部份Geobacillus菌株之16S rRNA基因序列具有極高之相似度，因此無法以目前常用之16S rRNA基因序列相似性來進行分類，本研究綜合多種目前常用的分子親緣特性分析方法，例如：胞內總蛋白SDS-PAGE圖譜、變性梯度膠體(denatrue gradient gel electrophoresis, DGGE)電泳圖譜、隨機擴增片段多形性DNA圖譜分析(randomly amplified polymorphic DNA analysis, RAPD)、多重基因座序列分析法(multilocus sequence analysis; MLSA)與脈衝式電泳(pulse field gel electrophoresis, PFGE)圖譜等技術進行Geobacillus屬內菌種之分群研究，以彌補16S rRNA基因序列於Geobacillus屬菌種親緣分析能力之不足。 本研究共使用17株Geobacillus參考菌株進行本屬分子親緣特性資料庫之建立。16S rRNA基因序列親緣性分析結果，可將Geobacillus屬菌種分為九大群，其中第VIb與VIc群菌株間相似性達99%以上，顯示欲利用16S rRNA序列進行菌種鑑定工作相當困難。16S rRNA基因序列DGGE分析技術無法滿足Geobacillus屬菌株鑑別之需求。胞內總蛋白SDS-PAGE圖譜相似性分析結果顯示’Bacillus caldotenax’ BCRC 11956、’B. caldovelox’ BCRC 11957為親緣極為相近之同一菌種，並與G. thermocatenulatus BCRC 17466、’B. caldolyticus’ BCRC 11954及G. kaustophilus BCRC 11223有相近的親緣關係。RAPD圖譜親緣性分析結果顯示‘B. caldotenax’ BCRC 11956與‘B. caldovelox’ BCRC 11957親緣相近；此外，G. kaustophilus BCRC 11223與‘B. caldolyticus’ BCRC 11954親緣相近。以引子對recA f1/r1及recA-f11/r11可專一地放大16株Geobacillus屬(第V、VIb與VIc群)參考菌株之recA基因片段，經Bioedit軟體與NCBI網站所提供的BLAST功能進行recA序列對齊分析，結果發現第V、VIb與VIc群中的10株參考菌株之recA序列可依是否含有內含子(intron)及其插入recA基因序列的位置可進一步區分為三種不同類型：即Type I、type II (II-1與II-2)及type III等。反轉錄聚合酶連鎖反應(reverse transcription PCR, RT-PCR)分析結果顯示不同Geobacillus屬菌株之cDNA樣本經引子對recA-f11/r11進行PCR放大後之產物分子量大小均相同，產物以引子對recA-f11/r11進行序列定序後，再分析其轉譯為胺基酸之序列結果完全相同。於實際應用上，可輕易地比較各菌株主要條帶之差異來判斷不同菌株所含之recA種類。rpoB基因序列相似度分析結果顯示對於Geobacillus屬中具有高度16S rRNA序列相似性之第VIb與VIc菌株而言可有較高之鑑別力。脈衝式電泳分析方面，使用HEPES取代0.5倍TEB做為PFGE之緩衝液可明顯改善基因體DNA被electrophoresis-related, Tris-dependent degradation的現象。並成功分析第VIc群嗜高溫菌屬Geobacillus參考菌株染色體DNA之脈衝電泳圖譜之差異。綜合上述分析之結果顯示，分類地位未定之‘B. caldolyticus’ BCRC 11954由於含有type II-2 recA基因，且RAPD與ropB等分析結果均支持其最相近之菌種為G. kaustophilus。另二株分類地位未定之‘B. caldotenax’ BCRC 11956、‘B. caldovelox’ BCRC 11957與G. vulcani BCRC 17563同樣均含有type III recA基因，且綜合胞內總蛋白SDS-PAGE圖譜、RAPD、rpoB與PFGE等分析數據皆顯示二者親緣極近。由上述之分析結果可知，部份Geobacillus菌株雖然具有極高的16S rRNA基因相似性，以目前著重16S rRNA基因序列組成為主要分類依據的體系無法滿足菌種鑑定之需求，但若能綜合應用RAPD、recA與rpoB等相關基因序列分析技術，則可有效解決具爭議的菌種分類議題。

Geobacillus species are predominantly found in “hot” environments such as compost or deep oil reservoirs. Some reports revealed the abilities of Geobacillus thermoleovorans on degradation of phenol compounds and the metabolic capabilities suggested that they may have important biotechnological applications, both in the industrial and environmental fields. Some Geobacillus species have highly similar 16S rRNA gene sequences, making 16S rDNA sequence analysis-based identification problematic. To overcome this limitation, different analysis methods including SDS-PAGE of whole-cell proteins, denature gradient gel electrophoresis (DGGE), randomly amplified polymorphic DNA (RAPD), recA and rpoB gene phylogenetic and PFGE analyses were evaluated as alternatives for distinguishing Geobacillus species. The phylogram of 16S rRNA gene sequences inferred from the neighbour-joining method showed that nine clusters of Geobacillus species were characterized with bootstrap values >90%. Cluster VIa (G. uzenensis and G. jurassicus) and VIb (eight strain of G. stearothermophilus) were supported by bootstrap values of 987 and 990, respectively. However cluster VIc harboured more diverse strains and had highly similar 16S rRNA gene sequences (>99.0%) and were indistinguishable by this methodology. The phylogenetic analysis of SDS-PAGE for whole-cell protein indicated that ’Bacillus caldotenax’ BCRC 11956 was closely related to ’B. caldovelox’ BCRC 11957, G. thermocatenulatus BCRC 17466, ’B. caldolyticus’ BCRC 11954 and G. kaustophilus BCRC 11223. The phylogenetic analysis of RAPD revealed that ‘B. caldotenax’ BCRC 11956 was related to ‘B. caldovelox’ BCRC 11957. On the other hand, G. kaustophilus BCRC 11223 was related to ‘B. caldolyticus’ BCRC 11954. Because 16S rRNA gene sequence analysis was inadequate for distinguishing the Geobacillus species in clusters VIb and VIc, similarity analysis of recA gene sequences was performed as an alternative. The primer set recA-f1/r1 was used to specifically amplify the partial recA gene (including the start codon) of 10 reference strains in clusters V, VIb, and VIc of Geobacillus; the resulting recA amplicons varied in length. We aligned the recA gene sequences of the 10 reference strains in clusters V, VIb, and VIc and found three different types of recA genes defined by whether they contained the intron and the location of the intron splice site in recA gene. The type I recA gene in G. thermodenitrificans BCRC 11733 had no intron. The type II recA gene had an insertion site GCAGAGCACGC GCTCGACCCC producing a recA-A fragment (214 bp in length) and a recA-B fragment (509 bp in length) with intron lengths of 1024 bp (type II-1) and 2783/2789 bp (type II-2), respectively. The type III recA gene had an insertion site GGCGAGCAAG/ CGCTCGAAATC producing a recA fragment (295 bp in length) and a recA-B fragment (428 bp in length) with introns length of 1528 bp and 1543 bp, respectively. Moreover, the putative amino acid sequences of recombinase-A encoded by different types of recA-A and recA-B genes in clusters V, VIb, and VIc were identical to each other. The primer set recA-f11/r11 (positions 177 to 739, GenBank accession number EU484369) was designed to amplify the recA gene from the most recently published and recognized strains of Geobacillus. All of the amplified RT-PCR products from the total RNA of 10 strains in clusters VIb and VIc of Geobacillus were identical to the PCR products from the genomic DNA of G. thermodenitrificans BCRC 11733 (positions 177 to 739, GenBank accession number EU484369). Phylogenetic analysis of rpoB gene sequences could effectively differentiate species in the VIb and VIc clusters that had highly similar 16S rRNA gene sequences. Electrophoresis-related, Tris-dependent degradation of G. thermoleovorans DNA was prevented by the use of HEPES buffer instead of 0.5X TBE buffer. The rapid procedures of PFGE and the program tool for restriction enzyme selection described in this study will make the successful PFGE typing of thermophilic Geobacillus species within 30-h period. In conclusion, according to the results of 16S rRNA, RAPD, recA, and rpoB gene sequences analyses, the controversial and uncategorized species ‘B. caldolyticus’ BCRC 11954 was assigned to the VIc cluster of Geobacillus and harboured a type II-2 recA gene, similar to G. kaustophilus BCRC 11223. However, ‘B. caldotenax’ BCRC 11956 and ‘B. caldovelox’ BCRC 11957 belonged to the VIc cluster of Geobacillus, had type III recA gene, and were related to G. vulcani BCRC 17563 according to the supports of SDS-PAGE, RAPD, rpoB and PFGE analysis results. Therefore, RAPD, recA and rpoB gene sequences analyses provided the rapid and reliable means for identifying Geobacillus species.