School of Natural Sciences, 14 Eastern Road, Macquarie University, Sydney, NSW, Australia.
ARC Centre of Excellence for Synthetic Biology, 14 Eastern Road, Macquarie University, Sydney, NSW, Australia.
J Antimicrob Chemother. 2024 Jan 3;79(1):100-111. doi: 10.1093/jac/dkad353.
Our study aimed to sequence class 1 integrons in uncultured environmental bacterial cells in freshwater from suburban creeks and uncover the taxonomy of their bacterial hosts. We also aimed to characterize integron gene cassettes with altered DNA sequences relative to those from databases or literature and identify key signatures of their molecular evolution.
We applied a single-cell fusion PCR-based technique-emulsion, paired isolation and concatenation PCR (epicPCR)-to link class 1 integron gene cassette arrays to the phylogenetic markers of their bacterial hosts. The levels of streptomycin resistance conferred by the WT and altered aadA5 and aadA11 gene cassettes that encode aminoglycoside (3″) adenylyltransferases were experimentally quantified in an Escherichia coli host.
Class 1 integron gene cassette arrays were detected in Alphaproteobacteria and Gammaproteobacteria hosts. A subset of three gene cassettes displayed signatures of molecular evolution, namely the gain of a regulatory 5'-untranslated region (5'-UTR), the loss of attC recombination sites between adjacent gene cassettes, and the invasion of a 5'-UTR by an IS element. Notably, our experimental testing of a novel variant of the aadA11 gene cassette demonstrated that gaining the observed 5'-UTR contributed to a 3-fold increase in the MIC of streptomycin relative to the ancestral reference gene cassette in E. coli.
Dissecting the observed signatures of molecular evolution of class 1 integrons allowed us to explain their effects on antibiotic resistance phenotypes, while identifying their bacterial hosts enabled us to make better inferences on the likely origins of novel gene cassettes and IS that invade known gene cassettes.
本研究旨在对来自郊区溪流淡水环境中未培养的细菌细胞中的 1 类整合子进行测序，揭示其细菌宿主的分类学信息。我们还旨在对与数据库或文献中序列不同的整合子基因盒进行特征描述，并确定其分子进化的关键特征。
我们应用了一种基于单细胞融合 PCR 的技术——乳液、配对分离和串联 PCR（epicPCR）——将 1 类整合子基因盒阵列与细菌宿主的系统发育标记物连接起来。实验中，在大肠杆菌宿主中定量测定了 WT 和改变的 aadA5 和 aadA11 基因盒编码的氨基糖苷（3"）腺苷酰转移酶赋予的链霉素抗性水平，这些基因盒编码的氨基糖苷（3"）腺苷酰转移酶。
在α变形菌和γ变形菌宿主中检测到 1 类整合子基因盒阵列。三个基因盒的子集显示出分子进化的特征，即获得了调节 5'非翻译区（5'-UTR），相邻基因盒之间 attC 重组位点的丢失，以及 IS 元件对 5'-UTR 的入侵。值得注意的是，我们对 aadA11 基因盒的一种新变体的实验测试表明，与大肠杆菌中祖先参考基因盒相比，获得观察到的 5'-UTR 导致链霉素 MIC 增加了 3 倍。
对 1 类整合子的分子进化观察特征进行剖析，使我们能够解释其对抗生素耐药表型的影响，同时鉴定其细菌宿主，使我们能够更好地推断新基因盒和入侵已知基因盒的 IS 的可能来源。
