Cady K C, White A S, Hammond J H, Abendroth M D, Karthikeyan R S G, Lalitha P, Zegans M E, O'Toole G A
1Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755, USA.
2Department of Ocular Microbiology, Aravind Eye Hospital, Madurai, India.
Microbiology (Reading). 2011 Feb;157(2):430-437. doi: 10.1099/mic.0.045732-0.
Here, we report the characterization of 122 Pseudomonas aeruginosa clinical isolates from three distinct geographical locations: Dartmouth Hitchcock Medical Center in New Hampshire, USA, the Charles T. Campbell Eye Microbiology Lab at the University of Pittsburgh Medical Center, USA, and the Aravind Eye Hospital in Madurai, India. We identified and located clustered regularly interspaced short palindromic repeats (CRISPR) in 45/122 clinical isolates and sequenced these CRISPR, finding that Yersinia subtype CRISPR regions (33 %) were more prevalent than the Escherichia CRISPR region subtype (6 %) in these P. aeruginosa clinical isolates. Further, we observed 132 unique spacers from these 45 CRISPR that are 100 % identical to prophages or sequenced temperate bacteriophage capable of becoming prophages. Most intriguingly, all of these 132 viral spacers matched to temperate bacteriophage/prophages capable of inserting into the host chromosome, but not to extrachromosomally replicating lytic P. aeruginosa bacteriophage. We next assessed the ability of the more prevalent Yersinia subtype CRISPR regions to mediate resistance to bacteriophage infection or lysogeny by deleting the entire CRISPR region from sequenced strain UCBPP-PA14 and six clinical isolates. We found no change in CRISPR-mediated resistance to bacteriophage infection or lysogeny rate even for CRISPR with spacers 100 % identical to a region of the infecting bacteriophage. Lastly, to show these CRISPR and cas genes were expressed and functional, we demonstrated production of small CRISPR RNAs. This work provides both the first examination to our knowledge of CRISPR regions within clinical P. aeruginosa isolates and a collection of defined CRISPR-positive and -negative strains for further CRISPR and cas gene studies.
在此，我们报告了从三个不同地理位置收集的122株铜绿假单胞菌临床分离株的特征：美国新罕布什尔州的达特茅斯希区柯克医疗中心、美国匹兹堡大学医学中心的查尔斯·T·坎贝尔眼微生物学实验室以及印度马杜赖的阿拉文德眼科医院。我们在122株临床分离株中的45株中鉴定并定位了成簇规律间隔短回文重复序列（CRISPR），并对这些CRISPR进行了测序，发现耶尔森氏菌亚型CRISPR区域（33%）在这些铜绿假单胞菌临床分离株中比大肠杆菌CRISPR区域亚型（6%）更为普遍。此外，我们从这45个CRISPR中观察到132个独特的间隔序列，它们与原噬菌体或已测序的能够成为原噬菌体的温和噬菌体100%相同。最有趣的是，所有这132个病毒间隔序列都与能够插入宿主染色体的温和噬菌体/原噬菌体匹配，但与染色体外复制的溶菌性铜绿假单胞菌噬菌体不匹配。接下来，我们通过从已测序的菌株UCBPP - PA14和六株临床分离株中删除整个CRISPR区域，评估了更普遍的耶尔森氏菌亚型CRISPR区域介导对噬菌体感染或溶原性的抗性的能力。我们发现，即使对于间隔序列与感染噬菌体区域100%相同的CRISPR，其介导的对噬菌体感染的抗性或溶原率也没有变化。最后，为了证明这些CRISPR和cas基因是表达且有功能的，我们展示了小CRISPR RNA的产生。这项工作不仅是我们所知的对临床铜绿假单胞菌分离株中CRISPR区域的首次研究，还提供了一组明确的CRISPR阳性和阴性菌株，用于进一步的CRISPR和cas基因研究。
