Matthysse A G, Yarnall H A, Young N
Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA.
J Bacteriol. 1996 Sep;178(17):5302-8. doi: 10.1128/jb.178.17.5302-5308.1996.
Transposon mutants of Agrobacterium tumefaciens which were avirulent and unable to attach to plant cells were isolated and described previously. A clone from a library of Agrobacterium tumefaciens DNA which was able to complement these chromosomal att mutants was identified. Tn3HoHo1 insertions in this clone were made and used to replace the wild-type genes in the bacterial chromosome by marker exchange. The resulting mutants were avirulent and showed either no or very much reduced attachment to carrot suspension culture cells. We sequenced a 10-kb region of this clone and found a putative operon containing nine open reading frames (ORFs) (attA1A2BCDEFGH). The second and third ORFs (attA2 and attB) showed homology to genes encoding the membrane-spanning proteins (potB and potH; potC and potI) of periplasmic binding protein-dependent (ABC) transport systems from gram-negative bacteria. The homology was strongest to proteins involved in the transport of spermidine and putrescine. The first and fifth ORFs (attA1 and attE) showed homology to the genes encoding ATP-binding proteins of these systems including potA, potG, and cysT from Escherichia coli; occP from A. tumefaciens; cysA from Synechococcus spp.; and ORF-C from an operon involved in the attachment of Campylobacte jejuni. The ability of mutants in these att genes to bind to host cells was restored by addition of conditioned medium during incubation of the bacteria with host cells.
先前已分离并描述了根癌土壤杆菌的转座子突变体，这些突变体无毒且无法附着于植物细胞。从根癌土壤杆菌DNA文库中鉴定出一个能够互补这些染色体附着突变体的克隆。在该克隆中进行了Tn3HoHo1插入，并通过标记交换用于取代细菌染色体中的野生型基因。所得突变体无毒，并且对胡萝卜悬浮培养细胞的附着能力要么没有，要么大大降低。我们对该克隆的一个10 kb区域进行了测序，发现一个推定的操纵子，其中包含九个开放阅读框（ORF）（attA1A2BCDEFGH）。第二个和第三个ORF（attA2和attB）与编码革兰氏阴性菌周质结合蛋白依赖性（ABC）转运系统的跨膜蛋白（potB和potH；potC和potI）的基因具有同源性。与参与亚精胺和腐胺转运的蛋白质的同源性最强。第一个和第五个ORF（attA1和attE）与编码这些系统的ATP结合蛋白的基因具有同源性，这些基因包括来自大肠杆菌的potA、potG和cysT；来自根癌土壤杆菌的occP；来自聚球藻属的cysA；以及来自参与空肠弯曲菌附着的操纵子的ORF-C。在细菌与宿主细胞孵育期间添加条件培养基可恢复这些att基因中突变体与宿主细胞结合的能力。
