Data_Sheet_1_Selective Isolation of Multidrug-Resistant Pedobacter spp., Producers of Novel Antibacterial Peptides.docx

Twenty-eight multidrug-resistant bacterial strains closely related or identical to Pedobacter cryoconitis, Pedobacter lusitanus and Pedobacter steynii were isolated from soil samples by selection for multidrug-resistance. Approximately 3–30% of the selected isolates were identified as Pedobacter, whereas isolation without antibiotics did not yield any isolates of this genus. Next generation sequencing data showed Pedobacter to be on 69th place among the bacterial genera (0.32% of bacterial sequences). The Pedobacter isolates produced a wide array of novel compounds when screened by UHPLC-MS/MSMS, and hierarchical cluster analysis resulted in several distinct clusters of compounds produced by specific isolates of Pedobacter, and most of these compounds were found to be peptides. The Pedobacter strain UP508 produced isopedopeptins, whereas another set of strains produced pedopeptins, which both are known cyclic lipodepsipeptides produced by Pedobacter sp. Other Pedobacter strains produced analogous peptides with a sequence variation. Further strains of Pedobacter produced additional novel antibacterial cyclic lipopeptides (ca 800 or 1400 Da in size) and/or linear lipopeptides (ca 700–960 Da in size). A 16S rRNA phylogenetic tree for the Pedobacter isolates revealed several distinct clades and subclades of isolates. One of the subclades comprised isolates producing isopedopeptin analogs, but the isopedopeptin producing isolate UP508 was clearly placed on a separate branch. We suggest that the non-ribosomal peptide synthases producing pedopeptins, isopedopeptins, and the analogous peptides, may derive from a common ancestral non-ribosomal peptide synthase gene cluster, which may have been subjected to a mutation leading to changed specificity in one of the modules and then to a modular rearrangement leading to the changed sequence found in the isopedopeptins produced by isolate UP508.

从土壤样本中，通过筛选多重耐药性，分离出二十八种与 Pedobacter cryoconitis、Pedobacter lusitanus 和 Pedobacter steynii 密切相关或完全相同的耐多药细菌菌株。在筛选的菌株中，约3-30%被鉴定为 Pedobacter，而未使用抗生素的分离过程中则未产生该属菌株。下一代测序数据显示，Pedobacter 在细菌属中排名第69位（细菌序列的0.32%）。通过超高效液相色谱-串联质谱/串联质谱（UHPLC-MS/MSMS）筛选，Pedobacter 菌株产生了多种新颖的化合物，系统聚类分析揭示了由特定 Pedobacter 菌株产生的多个独特的化合物簇，其中大部分化合物均为肽类。UP508 菌株产生的为 isopedopeptins，而另一组菌株则产生 pedopeptins，这两种化合物均为 Pedobacter sp. 产生的已知环状脂肽类糖肽。其他 Pedobacter 菌株产生了具有序列变异的类似肽类。进一步的研究中，Pedobacter 的某些菌株产生了额外的创新性抗菌环状脂肽（分子量约为800或1400 Da）和/或线性脂肽（分子量约为700-960 Da）。针对 Pedobacter 菌株的16S rRNA系统发育树揭示了多个独特的类群和亚类群。其中一个亚类群包括产生 isopedopeptin 类似物的菌株，但产生 isopedopeptin 的菌株 UP508 显然位于一个独立的分支上。我们推测，产生 pedopeptins、isopedopeptins 和类似肽类的非核糖体肽合成酶可能源自一个共同的祖先非核糖体肽合成酶基因簇，该基因簇可能经历了突变，导致其中一个模块的特异性改变，进而通过模块重组导致 UP508 菌株产生的 isopedopeptins 中发现的序列变化。