Holden P J, Brown R W
University of NSW, Kensington, Australia.
FEMS Microbiol Rev. 1993 Jul;11(1-3):19-30. doi: 10.1111/j.1574-6976.1993.tb00262.x.
Southern blot analysis of DNA from an iron-oxidising moderate thermophile NMW-6 and from Thiobacillus ferrooxidans strain TFI-35 demonstrated sequences homologous to the RuBisCO LSU gene of Synechococcus. DNA fragments (457 bp) encoding part of the RuBisCO LSU gene (amino acids 73-200) were amplified from the genomic DNA of Thiobacillus ferrooxidans and the moderate thermophile NMW-6 using the polymerase chain reaction (PCR) technique (Saiki et al. (1985) Science 233, 1350-1354). A comparison with the LSU sequences from T. ferrooxidans, Alcaligenes eutrophus, Chromatium vinosum, Synechococcus and Spinacea oleracea, which all have RuBisCOs with a hexadecameric structure, showed that the RuBisCO LSU gene sequence from NMW-6 appeared to be most closely related to that of the hydrogen bacterium A. eutrophus which showed 71.9% homology at the amino acid level. Despite its physiological similarity, T. ferrooxidans showed only 64.1% homology to the amino acid sequence from NMW-6 and had the lowest DNA homology (60.9%) of the hexadecameric type RuBisCOs. In the region sequenced, T. ferrooxidans and the RuBisCOs of the phototrophs C. vinosum, Synechococcus and S. oleracea, had 17 residues that were completely conserved which were substituted in both NMW-6 and A. eutrophus, 11 of these being identical substitutions. Comparison of the nucleotide and derived amino acid sequences of the RuBisCO LSU fragment from T. ferrooxidans with other RuBisCO sequences indicated a closer relationship to the hexadecameric type LSU genes of photosynthetic origin than to that of A. eutrophus. The T. ferrooxidans amino acid sequence showed 93.8%, 78.9% and 77.3% homology, respectively, to the C. vinosum, Synechococcus and S. oleracea (spinach) sequences but only 56.2% to A. eutrophus. The DNA sequence from Rhodospirillum rubrum, which has the atypical large subunit dimer RuBisCO structure with no small subunit, showed 39.2% and 42.7% homology, respectively, with the sequences of NMW-6 and T. ferrooxidans, and 25.0% and 29.7% amino acid homology, indicating that the DNA homology was substantially random in nature. PCR fragments (126 bp) that overlaped the last 15 codons of the fragments above were also amplified and sequenced. They showed incomplete homology with the larger fragments, supporting evidence obtained from Southern hybridizations that T. ferrooxidans and the moderate thermophile NMW-6 have multiple copies of RuBisCO LSU genes.
对铁氧化嗜中温菌NMW - 6和氧化亚铁硫杆菌菌株TFI - 35的DNA进行Southern印迹分析，结果显示存在与聚球藻属核酮糖-1,5-二磷酸羧化酶/加氧酶（RuBisCO）大亚基基因同源的序列。利用聚合酶链反应（PCR）技术（Saiki等人，（1985年）《科学》233卷，1350 - 1354页），从氧化亚铁硫杆菌和嗜中温菌NMW - 6的基因组DNA中扩增出编码RuBisCO大亚基基因部分序列（氨基酸73 - 200）的DNA片段（457 bp）。将其与来自氧化亚铁硫杆菌、真养产碱菌、嗜硫红假单胞菌、聚球藻属和菠菜的大亚基序列进行比较，这些物种的RuBisCO均具有十六聚体结构，结果表明NMW - 6的RuBisCO大亚基基因序列似乎与氢细菌真养产碱菌的序列关系最为密切，在氨基酸水平上具有71.9%的同源性。尽管氧化亚铁硫杆菌在生理特性上具有相似性，但其与NMW - 6的氨基酸序列同源性仅为64.1%，在具有十六聚体类型RuBisCO的物种中，其DNA同源性最低（60.9%）。在测序区域，氧化亚铁硫杆菌以及光合细菌嗜硫红假单胞菌、聚球藻属和菠菜的RuBisCO有17个完全保守的残基，在NMW - 6和真养产碱菌中这些残基被替换，其中11个是相同的替换。将氧化亚铁硫杆菌RuBisCO大亚基片段的核苷酸和推导的氨基酸序列与其他RuBisCO序列进行比较，结果表明其与光合起源的十六聚体类型大亚基基因的关系比与真养产碱菌的关系更为密切。氧化亚铁硫杆菌的氨基酸序列与嗜硫红假单胞菌（78.9%）、聚球藻属（77.3%）和菠菜（93.8%）的序列同源性分别为93.8%、78.9%和77.3%，但与真养产碱菌的同源性仅为56.2%。具有非典型大亚基二聚体RuBisCO结构且无小亚基的红螺菌的DNA序列与NMW - 6和氧化亚铁硫杆菌的序列同源性分别为39.2%和42.7%，氨基酸同源性分别为25.0%和29.7%，这表明DNA同源性在本质上基本是随机的。还扩增并测序了与上述片段最后15个密码子重叠的PCR片段（126 bp）。它们与较大片段显示出不完全同源性，这支持了Southern杂交获得的证据，即氧化亚铁硫杆菌和嗜中温菌NMW - 6具有多个RuBisCO大亚基基因拷贝。
