Krawczyk B, Lewandowski K, Kur J
Department of Microbiology, Technical University of Gdańsk, Poland.
Mol Cell Probes. 2002 Feb;16(1):1-11. doi: 10.1006/mcpr.2001.0388.
The recA gene is indispensable for a maintaining and diversification of the bacterial genetic material. Given its important role in ensuring cell viability, it is not surprising that the RecA protein is both ubiquitous and well conserved among a range of prokaryotes. Previously, we reported Acinetobacter genomic species identification method based on PCR amplification of an internal fragment of the recA gene with subsequent restriction analysis (RFLP) with HinfI and MboI enzymes. In present study, the PCR products containing the internal fragment of the recA gene, for 25 Acinetobacter strains belonging to all genomic species, were sequenced. Based on the nucleotide sequences the restriction maps and phylogenetic tree were prepared. The restriction maps revealed that Tsp509I restriction enzyme is the most discriminating for RFLP. To verify the computer analysis, the amplified DNAs from all reference genomic species available (43 strains) and 34 clinical strains were digested with each of the three restriction endonucleases mentioned. The results of digestion confirmed the computer analysis. The reconstructed phylogenetic tree showed linkages between genomic species 1 (Acinetobacter calcoaceticus), 2 (Acinetobacter baumannii), 3, 'between 1 and 3', TU13 and 'close to TU13'; genomic species 4, 6, BJ13, BJ14, BJ15, BJ16 and BJ17; genomic species 7 (Acinetobacter johnsonii) and TU14; genomic species 10 and 11; genomic species 8 (Acinetobacter Iwoffii), 9, 12 (Acinetobacter radioresistens) and TU15; and genomic species 5 (Acinetobacter junii). It is interesting that one branch in the phylogenetic tree contains haemolytic species-genomic species 4 (A. haemolyticus), BJ13, BJ14, BJ15, BJ16 and BJ17. The proposed genotypic method clearly revealed that the RFLP profiles obtained with Tsp509I enzyme might be useful for species identification of Acinetobacter strains. In this context, recA/RFLP genotypic method should be seen as an ideal preliminary screening method for large numbers of isolates, with the ultimate confirmatory role reserved for DNA hybridization analysis.
recA基因对于细菌遗传物质的维持和多样化是不可或缺的。鉴于其在确保细胞活力方面的重要作用，RecA蛋白在一系列原核生物中普遍存在且高度保守也就不足为奇了。此前，我们报道了基于recA基因内部片段的PCR扩增以及随后用HinfI和MboI酶进行限制性分析（RFLP）的不动杆菌基因组种鉴定方法。在本研究中，对属于所有基因组种的25株不动杆菌菌株的包含recA基因内部片段的PCR产物进行了测序。基于核苷酸序列制备了限制性图谱和系统发育树。限制性图谱显示Tsp509I限制性内切酶对RFLP的区分能力最强。为了验证计算机分析结果，用上述三种限制性内切酶分别消化了所有可用的参考基因组种（43株）和34株临床菌株的扩增DNA。消化结果证实了计算机分析结果。重建的系统发育树显示了基因组种1（醋酸钙不动杆菌）、2（鲍曼不动杆菌）、3、“在1和3之间”、TU13和“接近TU13”之间的联系；基因组种4、6、BJ13、BJ14、BJ15、BJ16和BJ17；基因组种7（约翰逊不动杆菌）和TU14；基因组种10和11；基因组种8（沃氏不动杆菌）、9、12（抗辐射不动杆菌）和TU15；以及基因组种5（琼氏不动杆菌）。有趣的是，系统发育树中的一个分支包含溶血种——基因组种4（溶血不动杆菌）、BJ13、BJ14、BJ15、BJ16和BJ17。所提出的基因型方法清楚地表明，用Tsp509I酶获得的RFLP图谱可能有助于不动杆菌菌株的种鉴定。在这种情况下，recA/RFLP基因型方法应被视为对大量分离株进行理想的初步筛选方法，而最终的确认作用则留给DNA杂交分析。
