O' Sullivan D, Ross R P, Twomey D P, Fitzgerald G F, Hill C, Coffey A
Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Ireland.
Appl Environ Microbiol. 2001 Feb;67(2):929-37. doi: 10.1128/AEM.67.2.929-937.2001.
The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (hsdS) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D. O'Sullivan, D. P. Twomey, A. Coffey, C. Hill, G. F. Fitzgerald, and R. P. Ross, Mol. Microbiol. 36:866-876; 2000). In this communication we provide evidence that the recombination event is favored by phage infection. The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization. The cadmium resistance property, encoded by the cadA gene, which has an associated regulatory gene (cadC), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation. In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria. The functionality of this intron was evident by demonstrating splicing in vivo. Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition.
噬菌体抗性质粒pAH90（26,490 bp）是一种天然共整合质粒，它通过两个较小的乳球菌质粒pAH33（6,159 bp）和pAH82（20,331 bp）的I型限制 - 修饰特异性决定簇（hsdS）之间的同源重组形成，在噬菌体攻击后产生噬菌体不敏感突变体（D.奥沙利文，D.P. 特沃米，A.科菲，C.希尔，G.F.菲茨杰拉德，和R.P.罗斯，《分子微生物学》36:866 - 876；2000年）。在本通讯中，我们提供证据表明重组事件受噬菌体感染的促进。测定了质粒pAH90的完整核苷酸序列，发现其包含24个开放阅读框（ORF），这些开放阅读框负责多种表型，包括限制 - 修饰、噬菌体吸附抑制、质粒复制、镉抗性、钴转运和接合转移。由cadA基因编码并带有相关调节基因（cadC）的镉抗性特性特别令人感兴趣，因为它有助于在电穿孔后在其他噬菌体敏感的乳球菌中选择pAH90。此外，我们报告鉴定了一个由两个外显子界定的II组自我剪接内含子，这两个外显子能够编码一种与革兰氏阳性菌接合有关的松弛酶。通过在体内证明剪接，该内含子的功能得以体现。鉴于pAH90编码在噬菌体裂解周期不同阶段起作用的强效噬菌体防御系统，这些系统与一个可在乳球菌之间转移的复制子上的食品级选择标记的联系，对于易受噬菌体抑制的工业乳制品发酵的天然菌株改良具有巨大潜力。
