| Summary: | 博士 === 國立臺灣大學 === 微生物與生化學研究所 === 97 === 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.
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