Division of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA.
J Bacteriol. 2014 Mar;196(6):1143-54. doi: 10.1128/JB.00739-13. Epub 2013 Dec 20.
In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.
为了更好地描述炭疽芽孢杆菌分型噬菌体 AP50c，我们设计了一个遗传筛选实验来鉴定其细菌受体。转座子 mariner 的插入或结构基因 S 层蛋白 Sap 和芽孢形成基因 spo0A、spo0B 和 spo0F 的靶向缺失，导致噬菌体抗性，同时伴随着噬菌体吸附和感染性缺陷。用 AP50c 孵育的细菌的电子显微镜显示，噬菌体颗粒与 Sterne 菌株杆菌的表面相关，但与 Δsap、Δspo0A、Δspo0B 或 Δspo0F 突变体的表面无关。与亲本菌株相比，每个 spo0 突变菌株的 S 层中 Sap 的量大大减少，并且将 AP50c 与纯化的重组 Sap 孵育会导致噬菌体活性大大降低。基于 B. cereus sensu lato 菌株全基因组序列的系统发育分析显示，有几个密切相关的 B. cereus 和 B. thuringiensis 菌株携带与炭疽芽孢杆菌 Sap 基因非常相似的 sap 基因。用野生型 B. anthracis sap 或对 AP50c 感染敏感的两种不同 B. cereus 菌株的 sap 基因对 Δsap 突变体进行反式互补，恢复了噬菌体敏感性，电子显微镜证实噬菌体颗粒附着在每个互补菌株的表面。基于这些数据，我们推测 Sap 参与 AP50c 的感染性，很可能作为噬菌体受体，而 spo0 基因可能调节 Sap 的合成和/或 S 层的形成。
