Suppression subtractive hybridization as a basis to assess Mycoplasma agalactiae and Mycoplasma bovis genomic diversity and species-specific sequences
Suppression subtractive hybridization as a basis to assess Mycoplasma agalactiae and Mycoplasma bovis genomic diversity and species-specific sequences
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DOI：
10.1099/mic.0.27590-0
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发表时间：
2005-02-01
期刊：
影响因子：
2.8
通讯作者：
Citti, C
中科院分区：
文献类型：
--
作者：
Marenda, MS;Sagné, E;Citti, C
系统发育相关的无乳支原体和牛支原体是两种难以区分的反刍动物病原体，其序列数据有限。为了评估这些支原体物种之间和物种内部存在的基因组多样性程度，通过抑制减法杂交分离出牛支原体型菌株PG45或无乳支原体型菌株PG2特异性DNA片段，并将其用作无乳支原体和牛支原体野外分离物的探针。结果表明，大约70%的特定类型菌株的DNA片段在所有的田间分离株中都有体现。只有一株牛分枝杆菌（最初被归类为无乳分枝杆菌）与15%的pg2特异性探针反应，而几株无乳分枝杆菌与15%的pg45特异性探针反应。序列分析表明，在一个物种内观察到的大部分基因组多样性与orf有关，这些orf与数据库中记录的蛋白质没有同源性或与限制性内切修饰系统编码的蛋白质同源性。让人想起基因转移作为基因组多样性的手段，在大多数牛支原体和少数无乳支原体分离物中发现了与发酵支原体（ICEF）整合偶联元件中心蛋白具有显著同源性的pg45特异性DNA片段。最后，在两组无乳乳杆菌和牛乳杆菌特异性DNA片段中发现了部分DNA聚合酶III的编码序列，并用于设计一种物种特异性PCR方法，用于鉴定和区分无乳乳杆菌和牛乳杆菌。
The phylogenically related Mycoplasma agalactiae and Mycoplasma bovis species are two ruminant pathogens difficult to differentiate and for which a limited amount of sequence data are available. To assess the degree of genomic diversity existing between and within these mycoplasma species, sets of DNA fragments specific for M. bovis type-strain PG45 or for M. agalactiae type-strain PG2 were isolated by suppression subtractive hybridization and used as probes on a panel of M. agalactiae and M. bovis field isolates. Results indicated that approximately 70% of the DNA fragments specific to one or the other type strain are represented in all field isolates of the corresponding species. Only one M. bovis isolate, which was first classified as M. agalactiae, reacted with 15% of the PG2-specific probes, while several M. agalactiae isolates reacted with 15% of the PG45-specific probes. Sequence analyses indicated that most of the genomic diversity observed within one species is related to ORFs with (i) no homologies to proteins recorded in the databases or (ii) homologies to proteins encoded by restriction modification systems. Reminiscent of gene transfer as a means for genomic diversity, a PG45-specific DNA fragment with significant homologies to a central protein of an integrative conjugative element of Mycoplasma fermentans (ICEF) was found in most M. bovis field isolates and in a few M. agalactiae isolates. Finally, sequences encoding part of DNA polymerase III were found in both sets of M. agalactiae- and M. bovis-specific DNA fragments and were used to design a species-specific PCR assay for the identification and differentiation of M. agalactiae and M. bovis.