Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A, spo0B, and spo0F in Phage AP50c Infection of Bacillus anthracis
Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A, spo0B, and spo0F in Phage AP50c Infection of Bacillus anthracis
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DOI：
10.1128/jb.00739-13
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发表时间：
2014-03-01
影响因子：
3.2
通讯作者：
Sozhamannan, Shanmuga
中科院分区：
文献类型：
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作者：
Plaut, Roger D.;Beaber, John W.;Sozhamannan, Shanmuga
为了更好地鉴定炭疽芽孢杆菌噬菌体AP50c的特性，我们设计了一种基因筛选器来鉴定其细菌受体。在炭疽杆菌中插入转座子水手或S层蛋白SAP结构基因和孢子形成基因sp0A、spo0B和spo0F的定向缺失导致噬菌体抗性，并伴随着噬菌体吸附和感染性的缺陷。用AP50c孵育的细菌的电子显微镜显示，噬菌体颗粒与Sterne菌株的细菌表面相关，而与Delta sap、Delta spo0A、Delta spo0B或Delta spo0F突变株的表面不相关。与亲本菌株相比，每株sp0突变株的S层中的SAP量都显著减少，AP50c与纯化的重组SAP孵育后，噬菌体活性显著降低。基于蜡状芽孢杆菌和苏云金芽孢杆菌全基因组序列的系统发育分析发现，几株与炭疽芽孢杆菌sap基因有很高相似性的菌株与蜡样芽孢杆菌和苏云金芽孢杆菌关系密切。用野生型炭疽杆菌突变株或两个对AP50c感染敏感的蜡状芽孢杆菌菌株的sap基因与Delta sap突变体的反式互补，恢复了噬菌体的敏感性，电子显微镜证实每个互补菌株的表面都附着了噬菌体颗粒。根据这些数据，我们推测SAP参与了AP50c的感染性，很可能是作为噬菌体受体，并且Spo0基因可能调节SAP的合成和/或S层的形成。
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 Delta sap, Delta spo0A, Delta spo0B, or Delta spo0F 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 wholegenome 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 Delta sap mutant in trans with the wildtype 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.