Stevenson D M, Kunnimalaiyaan M, Müller K, Vary P S
Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, 60115, USA.
Plasmid. 1998 Nov;40(3):175-89. doi: 10.1006/plas.1998.1359.
A replicon from one of an array of seven indigenous compatible plasmids of Bacillus megaterium QM B1551 has been cloned and sequenced. The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551. The cloned 2374-bp HindIII fragment was sequenced and contained two upstream palindromes and a large (>419-amino-acid) open reading frame (ORF) truncated at the 3' end. Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF. The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication. The ORF product was shown to act in trans. A small region with similarity to the B. subtilis chromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins. Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream. The predicted ORF protein of >48 kDa was basic and rich in glutamate + glutamine (16%). There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF. The data suggest that this is a theta replicon with an expressed rep gene required for replication. The replicon contains its iterons within the gene and has no homology to reported replicons. It is the first characterization of a B. megaterium replicon.
巨大芽孢杆菌QM B1551的七个本土相容质粒阵列中的一个复制子已被克隆并测序。该复制子与QM B1551菌株的所有四个大质粒（165、108、71和47 kb）杂交。对克隆的2374 bp HindIII片段进行了测序，其包含两个上游回文序列和一个在3'端截短的大的（>419个氨基酸）开放阅读框（ORF）。与大多数质粒起源不同，四个串联的12 bp直接重复序列区域位于该ORF内。单独的直接重复序列与复制子不相容，这表明它们是迭代子，并且该质粒可能通过θ复制进行复制。已证明ORF产物具有反式作用。检测到一个与枯草芽孢杆菌染色体起源膜结合区域相似的小区域，以及DnaA和IHF蛋白的可能结合位点。缺失分析表明最小复制子是一个1675 bp的片段，包含不完整的ORF加上上游的536 bp。预测的大于48 kDa的ORF蛋白呈碱性，富含谷氨酸+谷氨酰胺（16%）。与任何基因均无明显的氨基酸相似性，ORF中也不存在任何明显的基序。数据表明这是一个具有复制所需的表达rep基因的θ复制子。该复制子在基因内包含其迭代子，与已报道的复制子无同源性。这是巨大芽孢杆菌复制子的首次表征。
