Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA
J Bacteriol. 2018 Jun 11;200(13). doi: 10.1128/JB.00607-17. Print 2018 Jul 1.
The peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending in d-alanyl-d-lactate and binds with high affinity to peptidoglycan ending in d-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a native d-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genus Finally, our system represents the first "plug and play" counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium. The genus contains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selected species. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)-an essential enzyme involved in peptidoglycan synthesis-increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance in ; 81% of species are intrinsically resistant to vancomycin, which makes our tool broadly applicable.
乳酸菌的肽聚糖组成决定了万古霉素的耐药性。万古霉素与以 d-丙氨酰-d-乳酸结尾的肽聚糖结合能力相对较弱，与以 d-丙氨酰-d-丙氨酸（d-Ala-d-Ala）结尾的肽聚糖结合能力较强，分别导致万古霉素耐药性和敏感性。负责生成这些肽聚糖前体的酶是二肽连接酶（Ddl）。Ddl 活性位点中的一个氨基酸，苯丙氨酸或酪氨酸，分别决定了多肽或二肽的活性。在这里，我们通过异源表达万古霉素耐药乳酸菌中的二肽连接酶，建立了该酶在剂量依赖性方式下增加了其对万古霉素的敏感性，并克服了天然 d-Ala-d-Ala 二肽酶存在的影响。我们将二肽连接酶基因整合到自杀载体上，并证明其在乳酸菌中作为反向选择标记（CSM）发挥作用；万古霉素选择仅允许那些失去自杀载体的细胞生长。随后，我们开发了一种基于液体的方法，仅在 5 天内即可识别重组体，大约是传统方法所需时间的一半。系统发育分析表明，Ddl 可用作预测万古霉素耐药性的标记，从而表明 Ddl 作为反向选择标记在属中的广泛适用性。最后，我们的系统代表了第一个无需预先进行基因组编辑和/或合成培养基的“即插即用”反向选择系统。属中包含 200 多种物种，其中许多在食品和生物技术行业以及医学中被利用。目前，对固有万古霉素耐药性的预测仅限于选定的种。在这里，我们表明，肽聚糖合成中必需酶二肽连接酶（Ddl）的异源表达以剂量依赖性方式增加了对万古霉素的敏感性。我们利用这一点开发了一种反向选择标记物，用于万古霉素耐药性乳酸菌，从而扩展了目前大多数菌株可用的基因组编辑工具包。此外，我们还表明 Ddl 是一种可用于预测属中万古霉素耐药性的系统发育标记物；81%的种对万古霉素固有耐药，这使得我们的工具具有广泛的适用性。
