McGhee G C, Jones A L
Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824-1312, USA.
Appl Environ Microbiol. 2000 Nov;66(11):4897-907. doi: 10.1128/AEM.66.11.4897-4907.2000.
The complete sequence of plasmid pEA29 from Erwinia amylovora strain Ea88 consists of 28,185 bp with a 50.2% G+C content. As deletions and insertions were detected in other derivatives of pEA29, its size actually varied from 27.6 to 34.9 kb. Thirteen open reading frames that encoded predicted proteins with similarities to known proteins from other bacteria were identified along with two open reading frames related to hypothetical proteins found in GenBank and six open reading frames with no similarities to existing GenBank entries. Predicted products of open reading frames with similarity to the thiamine biosynthetic genes thiO, thiG, and thiF; a betT gene coding for choline transport; an msrA gene for the enzyme methionine sulfoxide reductase; a putative methyl-accepting chemotaxis gene; an aldehyde dehydrogenase gene; an hns DNA binding gene; a LysR-type transcriptional regulator; and parA and parB partitioning genes were identified. A putative iteron-containing theta-type origin of replication with an AT-rich region and a gene for a RepA protein was identified. PstI and KpnI restriction patterns for pEA29 isolated from tree fruit strains of E. amylovora were homogenous and different from those for pEA29 isolated from Rubus (raspberry) strains. All Rubus derivatives of pEA29 contained a point mutation that eliminated a PstI site and a 1,264-bp region that replaced 1, 890 bp of sequence found in pEA29 from strain Ea88. This change eliminated a second PstI site and increased the length of a KpnI fragment. An insertion sequence, ISEam1, was detected in one Rubus strain, and transposon Tn5393 was detected in three apple strains in two separate locations on the plasmid. Plasmid-cured strains exhibited reduced virulence and modified colony morphology on minimal medium without thiamine, indicating that some of the genes in pEA29 play a role in the physiology or metabolism of E. amylovora.
来自梨火疫病菌株Ea88的质粒pEA29的完整序列由28,185个碱基对组成，G+C含量为50.2%。由于在pEA29的其他衍生物中检测到缺失和插入，其大小实际上在27.6至34.9 kb之间变化。鉴定出13个开放阅读框，它们编码的预测蛋白质与其他细菌的已知蛋白质相似，同时还有两个与GenBank中发现的假设蛋白质相关的开放阅读框，以及六个与现有GenBank条目无相似性的开放阅读框。与硫胺素生物合成基因thiO、thiG和thiF相似的开放阅读框的预测产物；一个编码胆碱转运的betT基因；一个编码甲硫氨酸亚砜还原酶的msrA基因；一个假定的甲基接受趋化性基因；一个醛脱氢酶基因；一个hns DNA结合基因；一个LysR型转录调节因子；以及parA和parB分配基因。鉴定出一个假定的含迭代子的θ型复制起点，其具有富含AT的区域和一个RepA蛋白基因。从梨火疫病菌的树果菌株中分离出的pEA29的PstI和KpnI限制性酶切图谱是一致的，且与从悬钩子（覆盆子）菌株中分离出的pEA29的图谱不同。pEA29的所有悬钩子衍生物都含有一个点突变，该突变消除了一个PstI位点和一个1,264碱基对的区域，该区域取代了菌株Ea88的pEA29中发现的1,890碱基对的序列。这种变化消除了第二个PstI位点并增加了KpnI片段的长度。在一个悬钩子菌株中检测到一个插入序列ISEam1，在质粒上两个不同位置的三个苹果菌株中检测到转座子Tn5393。质粒消除菌株在不含硫胺素 的基本培养基上表现出毒力降低和菌落形态改变，这表明pEA29中的一些基因在梨火疫病菌的生理或代谢中起作用。
