Caufield P W, Shah G
University of Alabama at Birmingham, USA.
Dev Biol Stand. 1995;85:19-25.
Based on the observations reported here and what is known concerning transformation of naturally competent strains of S. mutans and other streptococcal species such as S. gordonii, we propose the model shown in Figure 2. The Tn916-intermediate transforms S. mutans as originally proposed for B. subtilis by Scott and coworkers [8]. It is not clear in either system (B. subtilis or S. mutans) whether the Tn916 intermediate enters the cell as ds-DNA or ss-DNA. Because it is likely that transformation of B. subtilis via formation of protoplasts involves a mechanism quite different from natural transformation in S. mutans, it would be unwise to extrapolate findings from their studies. If Tn916 enters S. mutans in a manner similar to plasmid or chromosomal DNA, we would assume that Tn916 binds to a cell receptor and as one strand enters, the other is degraded [9]. This leaves open the question of whether Tn916 recircularizes as ds-DNA before it inserts into the chromosome or whether it remains as ss-DNA, if, indeed, it enters as ss-DNA. The transformation efficiency for the Tn916 intermediate (approximately 10(-7) precluded kinetics studies such as those performed with pAM118. Poyart-Salmeron and coworkers [11] however, described a model in which Tn1545 inserts into the target site as a ds-DNA circular molecule, similar to that seen with lambda phage. Perhaps the most interesting finding presented here is that the predominant mechanism of insertion of Tn916 into the chromosome of the recipient occurs after Tn916 enters the cell. The replicative plasmid pAM118 evidently forms by two-hit kinetics followed by intracellular excision and transposition of Tn916. The helper-rescue experiment shows that in this system, the formation of Tcr transformants, and hence the integration of Tn916, was a function of the transformation efficacy of plasmid pAM118. Since intracellular excision of Tn916 probably follows the re-formation of the plasmid pAM118, the rate-limiting step in this system would be the formation of the transient, intracellular plasmid pAM118. (The transient white colony phenotype probably denotes the slower growth rate of transformants that acquire the large replicative plasmid pAM118). Our findings demonstrate that a practical way of promoting Tn916 insertions into chromosomal DNA for the purpose of obtaining mutations is to use a helper-rescue system. Our model supports the concept that the majority of Tn916 inserts arise from a mechanism similar to 'zygotic induction' as proposed for S. sanguis (gordonii) [1]. However, the frequencies for the co-establishment of the replicative plasmid (Emr) and Tn916 inserts (Tcr) in their paper (10(-6)) differ from our observations for S. mutans. We found nearly 100% of Tcr white colonies to be Emr whereas in S. gordonii, only a fraction (approximately 1%) exhibited the TcrEmr phenotype. If both phenotypes arose independently, the frequency of the TcrEmr phenotype would be 10(-8) rather than 10(-6) survivors/recipients as observed. It was surmised that both Tcr and Emr transformants arise dependently [1] where both the Tn916 intermediate and pAM118 contribute to the formation of Tn916 inserts. We conclude from their data, however, that most Tcr arose from the Tn916-intermediate formed in the donor, in agreement with the explanation of these data by Scott [12]. Support for the contention that the Tcr arose in S. gordonii from transformation by the Tn916-intermediate as in S. mutans (yellows), and not from intracellular excision from pAM118, comes from the observation that so few Tcr are Ems and because pAM150 (rep-) yields Tcr at the same frequency as the rep+ pAM118 in their experiments. In summary, the Tn916 intermediate is capable of transforming S. mutans. In contrast to the hypothesis of Scott [12], however, the Tn916 intermediate is not the only form involved in the transformat++t
基于此处报告的观察结果以及关于变形链球菌自然感受态菌株和其他链球菌物种（如戈登链球菌）转化的已知情况，我们提出了图2所示的模型。Tn916中间体转化变形链球菌的方式与Scott及其同事最初提出的枯草芽孢杆菌的方式相同[8]。在这两个系统（枯草芽孢杆菌或变形链球菌）中，尚不清楚Tn916中间体是以双链DNA还是单链DNA形式进入细胞。由于枯草芽孢杆菌通过原生质体形成进行的转化可能涉及与变形链球菌自然转化截然不同的机制，因此将他们研究中的发现进行外推是不明智的。如果Tn916以类似于质粒或染色体DNA的方式进入变形链球菌，我们会假设Tn916与细胞受体结合，当一条链进入时，另一条链被降解[9]。这就留下了一个问题，即Tn916在插入染色体之前是否会重新环化形成双链DNA，或者如果它确实以单链DNA形式进入，它是否会保持单链状态。Tn916中间体的转化效率（约10^(-7)）使得无法进行诸如用pAM118进行的动力学研究。然而，Poyart - Salmeron及其同事[11]描述了一种模型，其中Tn1545作为双链DNA环状分子插入靶位点，类似于λ噬菌体的情况。也许此处呈现的最有趣的发现是，Tn916插入受体染色体的主要机制发生在Tn916进入细胞之后。复制性质粒pAM118显然通过二次击中动力学形成，随后是Tn916的细胞内切除和转座。辅助拯救实验表明，在这个系统中，Tcr转化体的形成以及因此Tn916的整合是质粒pAM118转化效率的函数。由于Tn916的细胞内切除可能在质粒pAM118重新形成之后发生，这个系统中的限速步骤将是瞬时细胞内质粒pAM118的形成。（瞬时白色菌落表型可能表示获得大型复制性质粒pAM118的转化体生长速度较慢）。我们的发现表明，为了获得突变而促进Tn916插入染色体DNA的一种实用方法是使用辅助拯救系统。我们的模型支持这样的概念，即大多数Tn916插入来自类似于为血链球菌（戈登氏菌）提出的“合子诱导”的机制[1]。然而，他们论文中复制性质粒（Emr）和Tn916插入（Tcr）共同确立的频率（10^(-6)）与我们对变形链球菌的观察结果不同。我们发现几乎100%的Tcr白色菌落是Emr，而在戈登链球菌中，只有一小部分（约1%）表现出TcrEmr表型。如果两种表型独立出现，TcrEmr表型的频率将是10^(-8)，而不是观察到的10^(-6)存活者/受体。据推测，Tcr和Emr转化体都是依赖性产生的[1]，其中Tn916中间体和pAM118都有助于Tn916插入的形成。然而，我们从他们的数据得出结论，大多数Tcr来自供体中形成的Tn916中间体，这与Scott [12]对这些数据的解释一致。支持Tcr在戈登链球菌中是由Tn916中间体转化产生的观点（如同在变形链球菌中一样产生黄色菌落），而不是来自pAM118的细胞内切除，这一观点的证据来自于观察到很少有Tcr是Ems，并且因为在他们的实验中pAM150（rep-）产生Tcr的频率与rep+ pAM118相同。总之，Tn916中间体能够转化变形链球菌。然而，与Scott [12]的假设相反，Tn916中间体不是参与转化的唯一形式。
