The dipeptide D-Ala-D-Ala is an essential component of peptidoglycan and the target of vancomycin. Most Clostridioides difficile strains possess the vanG operon responsible for the synthesis of D-Ala-D-Ser, which can replace D-Ala-D-Ala in peptidoglycan. The C. difficile vanG operon is regulated by a two-component system, VanRS, but is not induced sufficiently by vancomycin to confer resistance to this antibiotic. Surprisingly, in the absence of the VanS histidine kinase (HK), the vanG operon is still induced by vancomycin and also by another antibiotic, ramoplanin, in a VanR-dependent manner. This suggested the cross-regulation of VanR by another HK or kinases that are activated in the presence of certain lipid II-targeting antibiotics. We identified these HKs as CD35990 and CD22880. However, mutations in either or both HKs did not affect the regulation of the vanG operon in wild-type cells suggesting that intact VanS prevents the cross-activation of VanR by non-cognate HKs. Overproduction of VanR in the absence of VanS, CD35990, and CD22880 led to high expression of the vanG operon indicating that VanR can potentially utilize at least one more phosphate donor for its activation. Candidate targets of CD35990- and CD22880-mediated regulation in the presence of vancomycin or ramoplanin were identified by RNA-Seq.

中文翻译：

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组氨酸激酶介导的艰难梭菌中万古霉素抗性操纵子的交叉调节


二肽 D-Ala-D-Ala 是肽聚糖的重要组成部分，也是万古霉素的靶标。大多数艰难梭菌菌株都具有负责合成D-Ala-D-Ser的vanG操纵子，它可以取代肽聚糖中的D-Ala-D-Ala。艰难梭菌 vanG 操纵子由双组分系统 VanRS 调节，但万古霉素不足以诱导其产生对该抗生素的抗性。令人惊讶的是，在没有 VanS 组氨酸激酶 (HK) 的情况下，vanG 操纵子仍然由万古霉素以及另一种抗生素雷莫拉宁以 VanR 依赖性方式诱导。这表明 VanR 受到另一种 HK 或激酶的交叉调节，这些 HK 或激酶在某些脂质 II 靶向抗生素存在时被激活。我们将这些 HK 识别为 CD35990 和 CD22880。然而，其中一个或两个 HK 的突变并不影响野生型细胞中 vanG 操纵子的调节，这表明完整的 VanS 可以防止非同源 HK 交叉激活 VanR。在没有 VanS、CD35990 和 CD22880 的情况下 VanR 的过量产生导致 vanG 操纵子的高表达，表明 VanR 可以潜在地利用至少一种磷酸供体来激活。通过 RNA-Seq 鉴定了万古霉素或雷莫拉宁存在下 CD35990 和 CD22880 介导的调节的候选靶标。