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Upon analysis of the<jats:named-content content-type=\"genus-species\">Legionella pneumophila<\/jats:named-content>strain 130b chromosome, we detected a subtype II-B CRISPR-Cas locus that contains<jats:italic>cas9<\/jats:italic>,<jats:italic>cas1<\/jats:italic>,<jats:italic>cas2<\/jats:italic>,<jats:italic>cas4<\/jats:italic>, and an array with 60 repeats and 58 unique spacers. Reverse transcription (RT)-PCR analysis demonstrated that the entire CRISPR-Cas locus is expressed during 130b extracellular growth in both rich and minimal media as well as during intracellular infection of macrophages and aquatic amoebae. Quantitative reverse transcription-PCR (RT-PCR) further showed that the levels of<jats:italic>cas<\/jats:italic>transcripts, especially those of<jats:italic>cas1<\/jats:italic>and<jats:italic>cas2<\/jats:italic>, are elevated during intracellular growth relative to exponential-phase growth in broth. Mutants lacking components of the CRISPR-Cas locus were made and found to grow normally in broth and on agar media.<jats:italic>cas9<\/jats:italic>,<jats:italic>cas1<\/jats:italic>,<jats:italic>cas4<\/jats:italic>, and CRISPR array mutants also grew normally in macrophages and amoebae. However,<jats:italic>cas2<\/jats:italic>mutants, although they grew typically in macrophages, were significantly impaired for infection of both<jats:italic>Hartmannella<\/jats:italic>and<jats:italic>Acanthamoeba<\/jats:italic>species. A complemented<jats:italic>cas2<\/jats:italic>mutant infected the amoebae at wild-type levels, confirming that<jats:italic>cas2<\/jats:italic>is required for intracellular infection of these host cells.<\/jats:p><jats:p><jats:bold>IMPORTANCE<\/jats:bold>Given that infection of amoebae is critical for<jats:named-content content-type=\"genus-species\">L. pneumophila<\/jats:named-content>persistence in water systems, our data indicate that<jats:italic>cas2<\/jats:italic>has a role in the transmission of Legionnaires\u2019 disease. Because our experiments were done in the absence of added phage, plasmid, or nucleic acid, the event that is facilitated by Cas2 is uniquely distinct from current dogma concerning CRISPR-Cas function.<\/jats:p>","DOI":"10.1128\/mbio.00074-13","type":"journal-article","created":{"date-parts":[[2013,3,13]],"date-time":"2013-03-13T03:31:48Z","timestamp":1363145508000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":95,"title":["The CRISPR-Associated Gene<i>cas2<\/i>of Legionella pneumophila Is Required for Intracellular Infection of Amoebae"],"prefix":"10.1128","volume":"4","author":[{"given":"Felizza F.","family":"Gunderson","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, Northwestern University, Medical School, Chicago, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas P.","family":"Cianciotto","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Northwestern University, Medical School, Chicago, Illinois, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-8-172"},{"key":"e_1_3_2_3_2","doi-asserted-by":"crossref","unstructured":"JansenR EmbdenJD GaastraW SchoulsLM . 2002. 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