Gregoire Stacy A, Byam Joel, Pavelka Martin S
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
Microbiology (Reading). 2017 Oct;163(10):1399-1408. doi: 10.1099/mic.0.000528.
is a fast-growing environmental organism and an important emerging pathogen. It is highly resistant to many antibiotics and undergoes a smooth to rough colony morphology change that appears to be important for pathogenesis. Smooth environmental strains have a glycopeptidolipid (GPL) on the surface, while certain types of clinical strains are often rough and lack this GPL, due to mutations in biosynthetic genes or the transporter gene. We report here the development and evaluation of an allelic exchange system for unmarked alleles in ATCC19977, using a suicide vector bearing the gene and 2-deoxygalactose counterselection. We describe here two variant suicide vectors, and demonstrate their utility in constructing a variety of mutants with deletion alleles of the GPL transporter gene, the GPL biosynthesis gene, the known β-lactamase gene and a putative β-lactamase gene, . We also show that a novel allele of the gene, conferring apramycin resistance (), can be used as a selectable marker in ATCC19977 at single copy.
是一种快速生长的环境微生物，也是一种重要的新兴病原体。它对许多抗生素具有高度抗性，并经历从光滑到粗糙的菌落形态变化，这似乎对发病机制很重要。光滑的环境菌株表面有糖肽脂（GPL），而某些类型的临床菌株通常是粗糙的且缺乏这种GPL，这是由于生物合成基因或转运基因发生突变所致。我们在此报告了一种用于在ATCC19977中对无标记等位基因进行等位基因交换系统的开发和评估，该系统使用携带基因和2-脱氧半乳糖反选择的自杀载体。我们在此描述了两种变体自杀载体，并证明了它们在构建具有GPL转运基因、GPL生物合成基因、已知β-内酰胺酶基因和推定β-内酰胺酶基因缺失等位基因的各种突变体中的效用。我们还表明，赋予安普霉素抗性（）的基因新等位基因可以在ATCC19977中作为单拷贝的选择标记使用。
