The CRISPR-Associated Gene cas2 of Legionella pneumophila Is Required for Intracellular Infection of Amoebae

Felizza F Gunderson; Nicholas P Cianciotto

doi:10.1128/mBio.00074-13

. 2013 Mar 12;4(2):e00074-13. doi: 10.1128/mBio.00074-13

The CRISPR-Associated Gene cas2 of Legionella pneumophila Is Required for Intracellular Infection of Amoebae

Felizza F Gunderson ¹, Nicholas P Cianciotto ¹

PMCID: PMC3604779 PMID: 23481601

ABSTRACT

Recent studies have shown that the clustered regularly interspaced palindromic repeats (CRISPR) array and its associated (cas) genes can play a key role in bacterial immunity against phage and plasmids. Upon analysis of the Legionella pneumophila strain 130b chromosome, we detected a subtype II-B CRISPR-Cas locus that contains cas9, cas1, cas2, cas4, 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 cas transcripts, especially those of cas1 and cas2, 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. cas9, cas1, cas4, and CRISPR array mutants also grew normally in macrophages and amoebae. However, cas2 mutants, although they grew typically in macrophages, were significantly impaired for infection of both Hartmannella and Acanthamoeba species. A complemented cas2 mutant infected the amoebae at wild-type levels, confirming that cas2 is required for intracellular infection of these host cells.

IMPORTANCE

Given that infection of amoebae is critical for L. pneumophila persistence in water systems, our data indicate that cas2 has a role in the transmission of Legionnaires’ 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.

Introduction

Clustered regularly interspaced palindromic repeat (CRISPR) arrays are found in ca. 40% of eubacteria and nearly all archaea (1–11). CRISPR arrays consist of palindromic repeats of 30 to 50 nt that are separated by unique spacers of 17 to 84 nt. Upstream of the array is an AT-rich leader that promotes transcription toward the repeats, and upstream of the leader are the CRISPR-associated sequence genes (cas genes) (2, 4, 12–14). Ten cas genes (cas1 to cas10) have been defined, although cas1 and cas2 are the only ones that are conserved in all bacteria (3, 11, 15–18). Based on the number and arrangement of cas genes, CRISPR loci are classified into types I, II, and III, which are further divided into 10 subtypes (A, B, C, etc.) (3, 15). In various bacteria, CRISPR and cas genes (CRISPR-Cas) have been linked to phage and plasmid immunity. CRISPR-Cas has also recently been shown to be capable of interfering with DNA transformation (28). CRISPR-mediated immunity occurs in three steps: adaptation, expression/maturation, and interference (3, 11, 16, 29, 30). In the first step, arrays acquire new spacers from the invading phage or plasmid, with acquisition occurring at the 5′ end of the array (19, 31). In the next step, upon the introduction of phage or plasmid DNA or RNA, the CRISPR array is transcribed, and then the long transcript is processed into smaller “crRNAs” by one or more Cas proteins (12, 32–37). In the interference step, the foreign DNA or RNA is recognized and cleaved by a Cas/crRNA complex (12, 38–42). In some cases, the activity of purified Cas proteins has been determined (11, 15); e.g., Cas1 is a DNA endonuclease involved in incorporation of spacers (43, 44), Cas2 is a site-specific endoribonuclease that is also implicated in spacer selection and/or integration (11, 45), Cas4 is a RecB-like DNA exonuclease linked to spacer acquisition (46, 47), and Cas9 is important for the production of crRNAs and cleaving target DNA (3, 48, 49). Reviews have challenged the field to take a broader look at CRISPR-Cas loci and examine their function in more bacteria (3, 6, 11, 17, 29, 50). Among eubacteria, ca. 12 types been experimentally examined for the physiologic role of the CRISPR-Cas locus (12, 19, 21, 22, 25–27, 34, 45, 48, 50–54), yet >400 bacteria have the locus (3). Also, ca. 98% of spacers do not have matches in GenBank, indicating that the origin of most spacers is unknown (46, 55). Finally, Takeuchi et al. have argued that Cas1 and Cas2 likely have roles that are distinct from and additional to their roles in phage and plasmid immunity (56). It was in this context that we embarked on the study of CRISPR-Cas in Legionella pneumophila.

L. pneumophila is a Gram-negative bacterium that causes a pneumonia known as Legionnaires’ disease (57, 58) that is of increasing incidence in the United States and elsewhere (59). Humans contract L. pneumophila by inhaling contaminated droplets that originate from aerosol-generating devices (58). In its aquatic habitats, L. pneumophila survives planktonically, as an intracellular parasite of protozoa, and in multiorganism biofilms (60–62). However, the major replicative niche of L. pneumophila is in amoebae (58, 61–63), with Acanthamoeba and Hartmannella species being the most critical hosts (60, 62–64). In lungs, L. pneumophila proliferates in macrophages (65, 66). Most sequenced strains have CRISPR arrays and cas genes (67, 68), and epidemiological studies indicate that a CRISPR-Cas locus is usually present in strains linked to disease (69). Previously examined L. pneumophila CRISPR spacers lack similarity to plasmid, phage, and other sequences in the database (68). No experiments on the role of CRISPR-Cas in L. pneumophila have been reported. We now demonstrate, among other things, that L. pneumophila cas genes are expressed upon intracellular infection and that the cas2 gene is required for infection of multiple amoebal hosts.

RESULTS

Detection of a CRISPR-Cas locus in L. pneumophila strain 130b.

Utilizing CRISPR Finder (70), we identified a single CRISPR-Cas locus in the chromosome of the virulent L. pneumophila strain 130b. The 130b CRISPR-Cas locus consisted of four cas genes (i.e., cas9, cas1, cas2, and cas4) and a downstream CRISPR array (Fig. 1). The positioning of the four cas genes suggested that they constitute an operon. Based upon the most recent CRISPR-Cas classification scheme, the 130b locus belongs to subtype II-B (3, 11, 15). A similar subtype II-B CRISPR-Cas locus was found in the chromosome and on a plasmid in L. pneumophila strain Paris, another clinical isolate belonging to serogroup 1 (67, 68). The amino acid sequences of the four 130b Cas proteins were 99 to 100% identical to those of the Paris Cas proteins. The direct repeats in the 130b CRISPR array were identical to the repeats in the Paris CRISPR array. However, the spacer sequences in strain 130b were different from those of strain Paris as well as being unlike any other sequences in the GenBank database. The 130b CRISPR-Cas locus was bounded by genes encoding hypothetical proteins (Fig. 1) and occupied a position in the bacterial chromosome location that was different that of the Paris chromosomal CRISPR array. To our knowledge, no members of CRISPR-Cas subtype II-B have been studied experimentally; the closest subtype to be examined is subtype II-A, which includes the loci of Streptococcus species (3, 15, 48, 71).

FIG 1 — The CRISPR-Cas locus of L. *pneumophila* strain 130b. Horizontal arrows denote the locations and orientations of *cas9*, *cas1*, *cas2*, and *cas4*, which are designated in the 130b genome as lpw_01781, lpw_01791, lpw_01801, and lpw_01811. Above the arrows, the sizes of the genes are indicated. There is a 3-bp overlap between *cas9* and *cas1*, a 7-bp gap between *cas1* and *cas2*, and a 60-bp gap between *cas2* and *cas4*. Located 216 bp downstream of the genes is a 4.2-kb CRISPR array which consists of 60 identical 37-bp direct repeats (grey diamonds) separated by 58 spacers that vary in size between 34 and 38 bp (black bars). The sequence of the repeats is indicated below the map. The CRISPR-Cas locus is bounded on one side by lpw_01771 and on the other by lpw_01861. There are 476 bp between the end of lpw_01771 and the start of *cas9* and 427 bp between the end of the CRISPR array and lpw_01861. The thin lines underneath signify the approximate sizes and locations of transcripts identified by RT-PCR.

Expression of the CRISPR-Cas locus in L. pneumophila grown extracellularly.

To begin to determine if the CRISPR-Cas locus is expressed by L. pneumophila grown under standard laboratory conditions, we grew strain 130b in buffered yeast extract (BYE) broth at 37°C (Fig. 2A) and then performed reverse transcription-PCR (RT-PCR) using primer pairs that are specific for each of the cas genes (Fig. 1). Transcripts corresponding to cas9, cas1, cas2, and cas4 were detected during exponential, early stationary, and late stationary phases (Fig. 2A). Additional RT-PCR analysis confirmed that the four cas genes are, as predicted, transcriptionally linked (see Fig. S1 in the supplemental material). cas genes were also expressed during growth at lower temperatures (Fig. 2B) as well as in a chemically defined medium (Fig. 2C). Pre-crRNA was also detected during growth in BYE broth at 25°C, 30°C, and 37°C and in cultures in chemically defined medium (CDM) (Fig. 2A to C). These data confirmed that all cas genes and pre-crRNA are expressed when L. pneumophila grows extracellularly. Using qRT-PCR, we next determined that the cas gene transcripts are more abundant in early stationary-phase cultures than they are in exponential-phase cultures (Fig. 2D). The cas2 gene was the most highly upregulated gene, displaying an approximately 75-fold-higher level of transcripts during stationary phase, compared to 30- to 45-fold elevations for cas9, cas1, and cas4. This type of expression profile, i.e., heightened expression during stationary phase, is reminiscent of the hyperexpression of infectious traits (72). This correlation suggested that the CRISPR-Cas locus might be relevant during intracellular infection by L. pneumophila.

FIG 2 — Expression of the *L. pneumophila* CRISPR-Cas locus during extracellular growth. (A, top panel) Growth of 130b in BYE broth. Strain 130b was inoculated into BYE broth and then incubated at 37°C. At various times postinoculation, the extent of growth was monitored spectrophotometrically. Data are means and standard deviations for triplicate cultures. The arrows indicate the points when samples were taken from exponential, early stationary, and late stationary phases for RNA extraction. (A, bottom panels) Expression of the CRISPR-Cas locus during exponential, early stationary, and late stationary phases in BYE broth at 37°C. RNA samples were analyzed by RT-PCR utilizing primers specific for *cas9*, *cas1*, *cas2*, *cas4*, or pre-crRNA. The amplicons obtained were resolved on agarose and visualized by ethidium bromide. As a control, amplification of 16S rRNA was included. That the PCR products obtained were from mRNA was confirmed by the lack of product obtained when the PCR mixture did not include RT. That the mRNAs were of the expected size was confirmed by comparing them to products obtained using genomic DNA. (B) Expression of the 130b CRISPR-Cas locus during early stationary phase in BYE broth at 25°C and 30°C, using the methods used for panel A. (C) Expression of 130b CRISPR-Cas during early stationary phase in CDM broth at 37°C, using the methods used for panel A. (D) The expression of *L. pneumophila* cas genes during early stationary phase compared to that during log phase. After 130b was grown in BYE broth at 37°C and bacterial RNA was obtained at exponential and early stationary phases, qRT-PCR and gene-specific primers were used to assess the fold change in *cas* gene expression during early stationary phase compared to exponential (E) phase. The data are means and standard deviations obtained from triplicate cultures or RNA samples. All of the increases in *cas* gene expression during the stationary phase were statistically significant, with the expression of *cas2* being significantly higher than that of the other three genes (P < 0.05; Student’s t test). All results are representative of three independent experiments.

Expression of the CRISPR-Cas locus in L. pneumophila grown intracellularly.

To determine if the L. pneumophila CRISPR-Cas locus is expressed during intracellular growth in macrophages, we infected human U937 cells with strain 130b (Fig. 3A) and then performed RT-PCR analysis on intracellular bacteria at 12, 18, 24, and 48 h after inoculation of the monolayer. These initial experiments demonstrated that cas9, cas1, cas2, cas4, and pre-crRNA are all expressed during growth in the macrophage cell line (Fig. 3B). That the transcripts were detected at 12 h and 18 h indicated that the CRISPR-Cas locus is expressed during the initial rounds of intracellular growth prior to lysis of the spent host cells. qRT-PCR analysis further demonstrated that the four cas genes are more highly expressed during intracellular infection (at 12 and 18 h) than they are during exponential growth in broth (Fig. 3C). cas1 and cas2 displayed the greatest elevation in gene expression during intracellular infection, with ca. 110-fold higher levels of mRNA at 12 h postinoculation and 40- to 70-fold higher levels at 18 h. Considering both time points, cas9 transcripts were elevated 10- to 30-fold and cas4 5- to 10-fold. To determine the CRISPR-Cas expression pattern in legionellae infecting a protozoan, we infected Acanthamoeba castellanii amoebae with 130b (Fig. 4A) and did additional RT-PCR analysis. Standard RT-PCR confirmed that cas9, cas1, cas2, cas4, and pre-crRNA are expressed during growth in acanthamoebae (see Fig. S2 in the supplemental material). Upon qRT-PCR analysis, the cas genes were again expressed at higher levels during intracellular infection than during broth growth (Fig. 4B). cas1 had the most notable increase in expression, exhibiting a ca. 100-fold increase in transcript levels. cas2 and cas9 transcripts were elevated ca. 25- and 50-fold, respectively, whereas cas4 transcripts were ca. 5-fold higher. These data confirmed that the CRISPR-Cas locus is expressed during infection of amoebae and macrophages, with the cas1 and cas2 genes exhibiting the most dramatic increases in expression when intracellular legionellae and extracellular legionellae are compared.

FIG 3 — Expression of the *L. pneumophila* CRISPR-Cas locus upon intracellular infection of macrophages. (A) Intracellular growth of 130b in macrophages. U937 cells were infected with 130b, and then at the indicated times, the numbers of CFU in the infected monolayers were determined. Data are means and standard deviations from three infected monolayers. Arrows indicate when samples were taken from the monolayers for RNA extraction. (B) Expression of the CRISPR-Cas locus at 12, 18, 24, and 48 h after inoculation. RNAs obtained from intracellular bacteria were analyzed by RT-PCR using primers specific for *cas9*, *cas1*, *cas2*, *cas4*, or pre-crRNA. That the PCR products obtained resulted from mRNA was confirmed by the lack of product obtained when the PCR did not use RT. (C) Expression of *L. pneumophila cas* genes during growth in macrophages compared to the log phase of extracellular growth. After 130b was grown in U937 cells at 37°C and bacterial RNA obtained at 12 and 18 h, qRT-PCR, and gene-specific primers were used to assess the fold change in *cas* gene expression during intracellular infection compared to the exponential (E) phase in BYE broth at 37°C. Data are means and standard deviations obtained from triplicate monolayers or RNA samples. All increases in *cas* gene expression during intracellular infection were statistically significant, with the expression of *cas1* and *cas2* being significantly higher than that of the other two *cas* genes at 12 h and the expression of *cas1* remaining higher at 18 h (P < 0.05; Student’s t test). All results are representative of three independent experiments.

FIG 4 — Expression of the *L. pneumophila* CRISPR-Cas locus during intracellular infection of *A. castellanii*. (A) Intracellular growth of 130b in *A. castellanii*. Amoebae were infected with 130b, and then at the indicated times, CFU in the cultures were determined. Data are means and standard deviations from four infected wells. The arrow indicates when samples were obtained for RNA extraction. (B) Expression of *cas* genes during intracellular growth in amoebae compared to the exponential phase of extracellular growth. After 130b was grown in amoebae at 37°C and RNA was obtained at 44 h postinoculation, qRT-PCR assessed the fold change in *cas* gene expression during infection compared to exponential (E) phase in BYE broth at 37°C. Data are means and standard deviations for four samples. All increases in expression during infection were significant, with the expression of *cas1* being significantly higher than that of the other genes (P < 0.05; Student’s t test). Data are representative of three experiments.

Isolation of L. pneumophila CRISPR-Cas mutants.

In order to determine if the CRISPR-Cas locus is needed for L. pneumophila growth, we generated a panel of 130b mutants specifically lacking cas1, cas2, cas4, cas9, or the CRISPR array. RT-PCR analysis determined that the mutation in each cas gene did not abolish the expression of the downstream cas gene(s) (data not shown). All of the mutants grew normally in BYE broth (see Fig. S3 in the supplemental material), indicating that the mutants do not have a generalized growth defect and that the Cas locus, though expressed, is not required for extracellular growth. The mutants exhibited typical colony morphology when grown on BCYE agar as well as normal shape and swimming motility (data not shown). They also behaved like the wild type did in terms of sliding motility, surfactant production, and secretion (see Fig. S4 in the supplemental material).

Intracellular infection by L. pneumophila CRISPR-Cas mutants.

To begin to determine the importance of CRISPR-Cas in infection, we assessed the relative ability of the mutants to grow in U937 cell macrophages. All of the mutants grew as well as the wild type did (see Fig. S5A to E in the supplemental material), indicating that cas1, cas2, cas4, cas9, and the CRISPR array are not required for optimal infection of macrophages. In support of this conclusion, all of the CRISPR-Cas mutants grew normally within bone marrow-derived (BMD) macrophages obtained from A/J mice (see Fig. S5F in the supplemental material). Turning to a protozoan model of intracellular infection, we observed that the cas1, cas4, cas9, and CRISPR array mutants grew normally in A. castellanii, indicating that Cas1, Cas4, Cas9, and the array are also not required for infection of protozoa (Fig. 5A, B, D, and E). In marked contrast, cas2 mutant NU411, although it grew normally in broth and in macrophages, exhibited significantly reduced recovery upon infection of the acanthamoebae (Fig. 5C). Indeed, at 48 h postinoc-ulation, the cas2 mutant-infected amoebal cultures contained about 55-fold fewer bacteria, and at 72 h, the cas2 mutant’s defect increased to approximately 1,000-fold. The cas2 mutant did not exhibit reduced survivability when incubated in the assay medium alone (data not shown), indicating that its reduced recovery from infected monolayers is due to impaired intracellular infection. Because a second, independently derived cas2 mutant (NU412) displayed the same defect (Fig. 6A), the reduced infectivity that we observed was likely due to the mutation of cas2 versus a spontaneous second-site mutation(s). Given that a mutation in cas4, the gene directly downstream of cas2, did not alter L. pneumophila infection of the amoebae (Fig. 6D), the reduced infectivity of the cas2 mutants was not due to a polar effect. Complementation of the mutant phenotype occurred when intact cas2 was introduced on a plasmid (Fig. 6B), confirming that the cas2 gene is required for optimal intracellular infection of A. castellanii. Because L. pneumophila is known to infect a variety of amoebae, we assessed the ability of the cas2 mutant and its complement to infect H. vermiformis. The cas2 mutant NU411, but not its complement, exhibited a reduced ability to infect the hartmannellae, displaying approximately 6-fold- and 20-fold-reduced recovery at 48 h and 72 h postinoculation, respectively (Fig. 6C). As a step toward possibly explaining the role of Cas2 in amoebae, we assessed the cas2 mutants’ sensitivity to DNA-damaging agents, because other Cas2 proteins are known to be nucleases (45, 73) and other bacterial Cas proteins have been implicated in resistance to DNA damage (74). However, the cas2 mutants did not show increased sensitivity to UV (see Fig. S6 in the supplemental material). Moreover, NU411 and NU412 did not display heightened sensitivity to mitomycin C or nalidixic acid; i.e., for both 130b and the mutants, the MIC for mitomycin C was 2.5 µg/ml, and that for nalidixic acid was 12.5 µg/ml. In sum, our infection data demonstrated that the cas2 gene and, by inference, the Cas2 protein are required for L. pneumophila intracellular infection of multiple amoebae.

FIG 5 — Intracellular growth of the wild type and *cas* mutants in *A. castellanii*. (A to E) Cultures of *A. castellanii* were infected with wild-type (WT) 130b, the *cas9* mutant NU409, the *cas1* mutant NU410, the *cas2* mutant NU411, the *cas4* mutant NU413, or the CRISPR array mutant NU414, and then at the indicated times, the numbers of CFU in the infected cultures were determined by plating. Data are means and standard deviations for four infected wells. Asterisks in panel C indicate points where the recovery of NU411 was significantly less than that of the wild type (P < 0.05; Student’s t test). Each panel is representative of at least three experiments.

FIG 6 — Intracellular growth of the wild type, *cas2* mutants, and a complemented *cas2* mutant in *A. castellanii* and *H. vermiformis*. (A to C) Monolayers of *A. castellanii* (A and B) and *H. vermiformis* (C) were infected with WT 130b, *cas2* mutant NU412, *cas2* mutant NU411, or complemented *cas2* mutant NU411 (pCas2), and at the indicated times, the numbers of CFU in the cultures were determined. Data are means and standard deviations for 4 infected monolayers. Asterisks indicate when the recovery of NU411 and NU412 was significantly less than that of WT and the complemented mutant (P < 0.05; Student’s t test). Each panel is representative of three independent experiments.

DISCUSSION

Arguably, one of the major findings in microbial genetics in recent years is the discovery of the CRISPR-Cas system and the characterization of its role in immunity against phage and plasmids. However, the possibility that a CRISPR-Cas system might do more than provide immunity to invading or transforming nucleic acid has only been hinted at. Here, we have demonstrated that the entire (subtype II-B) CRISPR-Cas locus of L. pneumophila is expressed under a wide variety of conditions, including extracellular replication in both rich and minimal media incubated at temperatures ranging from 25 to 37°C as well as intracellular multiplication in both mammalian macrophages and multiple aquatic amoebae. Importantly, qRT-PCR further documented that the levels of cas gene transcripts, especially those encoding Cas1 and Cas2, are appreciably greater during intracellular growth as well as in the late stationary phase of broth culture, which is known for the expression of infective traits. Even more significantly, we have found that the cas2 gene (and, by inference, the Cas2 protein) is required for the ability of L. pneumophila to optimally infect multiple types of amoebae. Because infection of protozoa is critical for L. pneumophila persistence in and transmission from man-made water systems, Cas2 must also have a significant role in the genesis of Legionnaires’ disease. For several reasons, we posit that the event that is mediated or facilitated by L. pneumophila Cas2 is entirely distinct from the current dogma concerning CRISPR-Cas function. First, although plasmids occur in some L. pneumophila strains and a preliminary study gives evidence for phage that can infect L. pneumophila (75, 76), our experiments were done in the absence of any added phage, plasmid, or nucleic acid. Second, only Cas2, not any other component of the L. pneumophila CRISPR-Cas locus, was required for infection. Our data add significantly to a growing set of observations that point toward there being other roles for the CRISPR-Cas locus. For example, Escherichia coli strains with deletions of cas1 or the array have increased sensitivity to DNA-damaging agents, suggesting that some components of the CRISPR-Cas system have a function in DNA repair (74). Moreover, envelope stress, in the absence of phage or plasmid, can activate transcription of E. coli CRISPR-Cas (77, 78). Compatible with these studies in E. coli, UV irradiation and osmotic stress increase cas gene transcription in Thermoproteus tenax (79). Finally, a CRISPR-Cas locus has been implicated in differentiation events in Myxococcus xanthus, the modulation of histidyl-tRNA pools in Pelobacter carbinolicus, and phage-dependent inhibition of biofilm formation by Pseudomonas aeruginosa (50, 52, 80–82). Our data are the first documentation of a novel role for cas2 or any component of a CRISPR-Cas locus in an infection event.

Members of the Cas2 family are small proteins (80 to 120 amino acids) that contain a ferredoxin fold that is found in RNA-binding proteins and an N-terminal β-strand followed by a polar amino acid, most often an aspartate or asparagine (45, 46, 83, 84). Cas2 proteins exist as homodimers (45). Crystal structures are known for six Cas2 proteins, including 3 from archaea (Sulfolobus solfataricus [2 paralogs] and Pyrococcus furiosus) and 3 from bacteria (Bacillus halodurans, Thermus thermophilus, and Desulfovibrio vulgaris) (11, 73, 84). The Cas2 proteins of 2 bacteria (Thermotoga maritime and Nitrosomonas europaea) and 3 archaea (S. solfataricus, Archaeoglobus fulgidus, and Methanobacterium thermoautotrophicum) have been shown to have endoribonuclease activity, cleaving single-stranded RNA preferentially within U-rich regions (45). Based on predicted structure and alignment to characterized Cas2 proteins, the Cas2 proteins from other eubacteria and archaea have been considered to be RNases (15, 46), although there is a recent report ascribing DNase activity to the Cas2 protein of B. halodurans (73). Using standard secondary-structure prediction programs, we determined that L. pneumophila Cas2 has both the N-terminal β-strand followed by aspartate and a ferredoxin fold analogous to those in B. halodurans, D. vulgaris, P. furiosus, S. solfataricus, and T. thermophilus (see Fig. S7 in the supplemental material). The 130b protein also has tyrosine, aspartic acids, and phenylalanine residues that are like those that were defined in S. solfataricus as being key for catalytic activity (45) (see Fig. S7). Thus, L. pneumophila Cas2 is probably an RNase or alternately a DNase. Because deletion of the entire CRISPR array did not decrease infectivity in the way that loss of cas2 did, it is unlikely that the processing of pre-crRNA by Cas2 is the critical event in infection of amoebae. Rather, it is conceivable that RNase activity modulates the level(s) or configuration(s) of another regulatory RNA(s) and/or mRNA(s) that influences or encodes factors needed for infection. In light of in vitro observations made with other bacteria, noted above, perhaps L. pneumophila Cas2 modulates RNAs that influence bacterial resistance to oxidative stress or other damaging agents that are present during infection of an amoebal host. That a bacterial cytoplasmic RNase could have this sort of effect is evident in the literature; e.g., the CvfA protein of Streptococcus pyogenes is an RNase with endonuclease activity that positively regulates virulence determinants (85). Clearly, further experimentation will need to be done in order to discern if L. pneumophila Cas2 is in fact an RNase (or DNase) and then ascertain if and how such a nuclease is necessary for optimal intracellular infection of host cells.

Given that the CRISPR-Cas locus of L. pneumophila 130b is essentially the same as that of L. pneumophila Paris, with the Cas2 proteins sharing 100% identity (see Fig. S7 in the supplemental material), our observations concerning the hyperexpression of CRISPR-Cas and the importance of Cas2 likely have implications for strain Paris as well as the many virulent serogroup 1 strains that are related to 130b and Paris (69, 86). Compatible with this hypothesis, a supplemental table included as part of a microarray study indicated that cas1, cas2, cas4, and cas9 of Paris are expressed during infection of A. castellanii (87). Because some strains, including Philadelphia-1 and Corby, do not carry a CRISPR-Cas locus (67, 68), Cas2 must not be an absolute requirement for intracellular infection by all strains of L. pneumophila. That strains can differ in terms of individual virulence determinants is not unheard of; e.g., there is variability among the effectors that are secreted via the type IV secretion system (57, 86). Finally, our results may also have implications for other CRISPR-Cas-containing bacteria, including other pathogens. For example, the subtype II-B locus of 130b is similar to an uncharacterized locus in Francisella tularensis strains (e.g., NCBI YP 898402.1 to 898405.1), with the predicted proteins sharing 42% amino acid identity (E value = 8 × 10⁻¹²); Francisella, like Legionella, can grow in amoebae (88).

In conclusion, we have demonstrated, among other things, that the L. pneumophila CRISPR-Cas locus is expressed during intracellular growth and that the cas2 gene in particular is required for infection of multiple types of amoebae. In addition to revealing the importance of a CRISPR-Cas component in the natural history and pathogenesis of Legionnaires’ disease, these results clearly indicate that L. pneumophila cas2 has a role that is unique from the prevailing view of CRISPR-Cas function. Our findings with Legionella further suggest that CRISPR-Cas loci present in other bacteria and archaea may have physiologically relevant functions that are unrelated to phage and plasmid immunity or DNA transformation and other forms of horizontal gene transfer.

MATERIALS AND METHODS

Bacterial strains, media, and extracellular growth assessments.

L. pneumophila serogroup-1 strain 130b (ATCC BAA-74) served as our wild type (89). Mutants that were isolated in this study are listed in Table 1. Legionellae were grown at 37°C on buffered charcoal yeast extract (BCYE) agar, which, when appropriate, contained chloramphenicol at 3 µg/ml, kanamycin at 25 µg/ml, or gentamicin at 2.5 µg/ml (89). Escherichia coli strain DH5α (Invitrogen, Carlsbad, CA) was the host for recombinant plasmids and was grown in Luria-Bertani medium. Unless otherwise noted, chemicals were from Sigma-Aldrich (St. Louis, MO). To monitor extracellular growth of L. pneumophila strains as well isolate RNA, legionellae were inoculated into buffered yeast extract (BYE) or chemically defined medium (CDM) broth and then incubated with shaking (90). The optical densities of cultures were determined at 660 nm (Beckman Coulter, Indianapolis, IN). Microscopy was used to observe shape and swimming motility. Sliding motility and surfactant production were monitored as previously described (89).

TABLE 1.

L. pneumophila strains used in this study

Strain(s)	Description	Reference or source
130b	Clinical isolate	89
NU409	cas9 mutant of strain 130b	This study
NU410	cas1 mutant of strain 130b	This study
NU411, NU412	cas2 mutants of strain 130b	This study
NU413	cas4 mutant of strain 130b	This study
NU414	CRISPR mutant of strain 130b	This study

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DNA and protein sequence analysis.

DNA was isolated from L. pneumophila as described before (89). Primers used for sequencing and/or PCR were obtained from Integrated DNA Technologies (Coralville, IA). Primer sequences are listed in Table S1 in the supplemental material. DNA sequences were analyzed using Lasergene (DNASTAR, Madison, WI), and protein alignments were done using the Clustal method. The CRISPRFinder software available at http://crispr.u-psud.fr/Server/ (70) was used to both identify the CRISPR-Cas locus (http://www.ncbi.nlm.nih.gov/nuccore/FR687201.1) and to analyze the individual spacers and repeats. Other BLAST homology searches were done through the National Center for Biotechnology Information (NCBI) and the other L. pneumophila databases at http://genolist.pasteur.fr/LegioList/. To obtain secondary structures, we used the I-TASSER and Phyre servers (90, 91, 92).

RT-PCR analysis.

To monitor L. pneumophila transcription, reverse transcription-PCR (RT-PCR) was done essentially as described before (90). RNA was isolated from BYE and CDM cultures by using the RNA STAT-60 reagent (Tel-Test, Friendswood, TX) and following the manufacturer’s instructions, with the exception that glycogen and sodium acetate were added during precipitation (93). To isolate bacterial RNA from infected host cells, U937 cells and Acanthamoeba castellanii were infected as described below. The monolayer was lysed with 50% RNA Protect (Qiagen, Valencia, CA)-1% saponin, and RNA was extracted using RNA STAT-60. RNA samples were treated with DNase I (Life Technologies, Carlsbad, CA), extracted using acid-phenol-chloroform, and precipitated with sodium acetate-ethanol (94). cDNA was synthesized in a 20-µl reaction mixture containing 1 µg of RNA, a 1 µM concentration of the gene-specific primer Cas9-4RT-R116 or 0.12 µg of random primers, and the following items obtained from Life Technologies: 1× first strand buffer, a 2 mM concentration of each deoxynucleoside triphosphate (dNTP), 10 mM dithiothreitol (DTT), 40 U RNaseOut, and 200 U of SuperScript III reverse transcriptase. Primers CAS1-F692 and CAS1-R869 were used to examine transcription of cas1, CAS2-F50 and CAS2-R293 were used for cas2, CAS4-F36 and CAS4-R279 were used for cas4, CAS9-F3047 and CAS9-R3188 were used for cas9, and CRISPRII-FL1 and CRISPRII-R5′.S5 were used for pre-crRNA transcripts. As a control, 16S rRNA gene transcription was assessed using primers 16S rRNA-F and 16S rRNA-R. In order to assess cotranscription of cas9 and cas1, primers CAS9-F3047 and CAS1-R282 were used. For monitoring cotranscription of cas1 and cas2, we used CAS1-F692 and CAS2-R293, and for cas2 and cas4, CAS2-F50 and CAS4-R279. Endpoint PCRs were separated by electrophoresis and detected with ethidium bromide.

Quantitative RT-PCR.

To precisely determine levels of RNA, quantitative RT-PCR (qRT-PCR) was done. The cDNA sample was diluted 1:20, and a 5-µl aliquot was added to a 25-µl reaction mixture, which included 12.5 µl of SYBR green master mix (Bio-Rad, Hercules, CA) and a 0.5 µM concentrated of each primer. The primer pairs used were as follows; CAS9-F3047 and CAS9-R3188 for cas9, Cas1-F and CAS1-R for cas1, CAS2-F and CAS2-R for cas2, and CAS4-F and CAS4-R for cas4. Using the CFX96 Touch real-time PCR detection system (Bio-Rad), the reaction cycle was 95°C for 3 min, 95°C for 10 s, and 60°C for 30 s for 40 cycles. For each assessment, three biological replicates were obtained, with each one being tested in triplicate. To quantitate products, standard curves using genomic DNA were generated for each primer set. L. pneumophila lpw_00031 (i.e., gyrB) and lpw_16991 were used as reference genes to normalize gene expression, as previously done (94–96). The primer pair for gyrB was GYRB-F and GYRB-R, and that for lpw_16991 was LPW16991-F and LPW16991-R. The level of gene expression was assessed by determining the cycle at which the amplification curve crossed the detection threshold, and the relative change in gene expression was calculated using the 2^ΔΔCT method (97).

Mutant construction and genetic complementation.

To generate L. pneumophila mutants lacking either cas9, cas1, cas2, cas4, or the CRISPR array, we performed variations on allelic exchange (89, 90). To obtain a cas9 (lpw_01781) mutant, the 5′ and 3′ ends of the gene were separately amplified from 130b DNA using primers CAS9-F1 and CAS9-R1SmaI and primers CAS9-F2SmaI and CAS9-R2, respectively. The generated fragments were ligated into pGEM-T Easy (Promega, Madison, WI), and the resulting plasmids were digested with SmaI and SpeI. Finally, a trimolecular ligation was done, placing a kanamycin resistance cassette (Km^r), obtained from pMB2190 (90), between the beginning and end of cas9. The plasmid obtained, pGEM-cas9-Km, had a 4.1-kb deletion in the center of cas9. Mutated cas9 was introduced into the chromosome of 130b by transformation (90) of pGEM-cas9-Km, and the mutant genotype was confirmed by PCR using primers CAS9-F19 and CAS9-R4079. Utilizing primers CAS1-F1 and CAS1-R1StuI and CAS1-F2StuI and CAS1-R2, a similar allelic exchange procedure was used to mutate cas1 (lpw_01791). In this case, the two initial plasmids were digested with StuI and SphI, and the trimolecular ligation inserted a gentamicin resistance cassette (Gm^r), obtained from pX1918-GT (90), in cas1. The final plasmid, pGEM-cas1-Gm, had an 800-bp deletion in cas1. Following transformation of pGEM-cas1-Gm into 130b, the genotype was confirmed by PCR using CAS1-F1 and CAS1-R2. To mutate cas2 (lpw_01801), CAS2-F1–CAS2-R1StuI and CAS2-F2StuI–CAS2-R2 were used, and the trimolecular ligation placed Km^r into cas2. The final plasmid made, pGEM-cas2-Km, had a 200-bp deletion in cas2. After transformation of pGEM-cas2-Km into 130b, the mutant was confirmed with primers CAS2-F1 and CAS2-R2. To obtain a cas4 (lpw_01811) mutant, allelic exchange was again used but with primer pairs CAS4-F1 and CAS4-R1StuI and CAS4-F2StuI and CAS4-R2. The initial pGEM-based plasmids were digested with StuI and SpeI, and the trimolecular ligation inserted Gm^r between the beginning and end of cas4. The final plasmid, pGEM-cas4-Gm, had a 350-bp deletion in cas4, and a mutated gene was confirmed using CAS4-F1 and CAS4-R2. To obtain a mutant that lacked the entire CRISPR array, the 5′ and 3′ ends of the array were separately amplified from 130b DNA using CRISPR-F1 and CRISPR-R1SmaI and CRISPR-F2SmaI and CRISPR-R2, respectively. The generated fragments was ligated into pGEM-T Easy, and the resulting plasmids were digested with SmaI and SpeI. Lastly, a trimolecular ligation was done by placing Km^r between the beginning and end of the array. The plasmid obtained, pGEM-CRISPR-Km, contained a 4.2-kb deletion. The mutated array was introduced into 130b by transformation, and the genotype was verified by PCR using primers CRISPR-DIAGF and CRISPR-DAIGR. To generate a plasmid for complementation of the cas2 mutant, intact cas2 (but no other gene) was amplified from 130b DNA using CAS2-SphI-F and CAS2-SacI-R. After the product was digested with SphI and SacI, the cas2-containing fragment was cloned into pMMB2002 (90), and the new pCas2 was electroporated (90) into mutant NU411.

Intracellular infection assays.

Hartmannella vermiformis (ATCC 50237) and A. castellanii (ATCC 30234) were infected with L. pneumophila as described before (89). To assess L. pneumophila growth in mammalian cells, we infected human U937 cells (ATCC CRL-1593.2) and bone marrow-derived (BMD) macrophages obtained from mice (98). To prepare the murine macrophages, progenitor cells were extracted from the femurs of A/J mice (The Jackson Laboratory, Bar Harbor, ME), laid down in plates, and incubated in RPMI medium with 20% fetal bovine serum (FBS), 30% L-cell supernatant, 1% penicillin-streptomycin, and 1 µg/ml Fungizone. On day 3, the cultures were given fresh medium. On day 7, cultures were washed with phosphate-buffered saline (PBS) and incubated at 4°C until the adherent cells detached. Recovered macrophages were used to form monolayers of 2.5 × 10⁵ cells, and after 3 days in RPMI with 10% FBS and 10% L-cell supernatant, monolayers were infected with bacteria as before (98).

Assays for bacterial sensitivity to DNA-damage.

To judge sensitivity to UV light, overnight BYE cultures of wild-type and mutant bacteria were diluted in BYE broth to an optical density at 660 nm (OD₆₆₀) equal to 0.3, and then 10-fold serial dilutions were made in PBS. Analogous to previous studies (99–102), we spotted 10-µl aliquots of the suspensions onto BCYE agar, and then the plates were exposed to various doses of 254-nm UV light (Stratalinker; Stratagene, Santa Clara, CA). To assess sensitivity to chemical damaging agents, we determined the MICs of mitomycin C and nalidixic acid according to a standard protocol using broth microdilutions (103–105). All sensitivity assays were performed on three independent occasions.

SUPPLEMENTAL MATERIAL

Figure S1

The L. pneumophila strain 130b cas operon. (Top) Depiction of the region of the 130b chromosome containing cas genes. The horizontal gray arrows denote the location and orientation of cas9, cas1, cas2, and cas4, which are also designated in the genome as lpw_01781, lpw_01791, lpw_01801, and lpw_01811, respectively. The thinner black arrow represents the primer used to generate gene-specific cDNA. The thin lines above the genes denote the approximate size and location of the three transcripts (P1, P2, and P3) identified by RT-PCR analysis; P1 amplified the junction between cas9 and cas1, P2 the junction between cas1 and cas2, and P3 the junction between cas2 and cas4. (Bottom) Detection of cas operon transcripts. RNA samples obtained from 37°C BYE cultures were analyzed by RT-PCR using primers specific for cas9, cas1, cas2, or cas4. The amplicons were resolved on an agarose gel and visualized by staining. That the PCR products were from mRNA templates was confirmed by the lack of product obtained when the PCR mixture did not include RT. That the mRNAs were of the expected size was confirmed when they were compared to products obtained using genomic DNA (gDNA). The leftmost lane in each panel is a 1-kb ladder of DNA fragments. Download

Figure S1, EPS file, 2.7 MB^{(2.7MB, eps)}

Figure S2

Expression of the L. pneumophila CRISPR-Cas locus during infection of A. castellanii. After strain 130b was grown in amoebae at 37°C and bacterial RNA obtained at 44 h postinoculation, RT-PCR and gene-specific primers were used to assess transcription. The amplicons obtained were resolved on an agarose gel and visualized by staining. As a control, amplification of 16S rRNA was included. That the products obtained were from mRNA templates was confirmed by the lack of product obtained when the PCR mixture did not include RT. That the mRNAs seen were of the expected size was confirmed when they were compared to products obtained using genomic DNA. The data are representative of three independent experiments. Download

Figure S2, EPS file, 2.2 MB^{(2.2MB, eps)}

Figure S3

Extracellular growth of L. pneumophila wild type and CRISPR-Cas mutants in bacteriological media. Growth of legionellae in BYE broth at 37°C. Wild-type 130b (●), the cas1 mutant NU410 (△), the cas2 mutant NU411 (○), and the cas4 mutant NN413 (◊) (A) or wild-type 130b (●), the cas9 mutant NU409 (□), and the CRISPR mutant NU414 (*) (B) were inoculated into BYE broth, and at various times postinoculation, growth was monitored spectrophotometrically. The data are means and standard deviations for triplicate cultures. All results are representative of two independent experiments. Download

Figure S3, EPS file, 0.4 MB^{(453.4KB, eps)}

Figure S4

Surface translocation by L. pneumophila CRISPR-Cas mutants. Wild-type strain 130b (A), the cas9 mutant NU409 (B), the cas1 mutant NU410 (C), the cas2 mutant NU411 (D), the cas4 mutant NU413 (E), and the CRISPR array mutant NU414 (F) were spotted onto BCYE plates containing 0.5% agar and then incubated at 30°C. After six days of incubation, the legionellae spread over the agar surface in an amorphous fashion, with a surfactant film advancing ahead of the growing bacteria. The wrinkled appearance of the agar surface in panel F was a peculiarity and is not associated with either the sliding phenotype or the NU414 mutant. Otherwise, the images presented are representative of those obtained from two independent experiments. Download

Figure S4, EPS file, 19.6 MB^{(19.6MB, eps)}

Figure S5

Intracellular growth of L. pneumophila wild type and cas mutants in macrophages (A to F). U937 cell macrophages (A to E) and BMD macrophages from A/J mice (F) were infected with either wild-type (WT) strain 130b (●), the cas9 mutant NU409 (□), the cas1 mutant NU410 (△), the cas2 mutant NU411 (○), the cas4 mutant NU413, or the CRISPR array mutant NU414 (_*), and then at the indicated times, the numbers of CFU in the infected monolayers were determined by plating. Data are means and standard deviations for three infected wells. Results in panels A to E are each representative of three independent experiments, and those in panel F are representative of two independent trials. Download

Figure S5, EPS file, 0.5 MB^{(503.9KB, eps)}

Figure S6

UV-sensitivity of L. pneumophila wild type and cas2 mutants. Ten-microliter aliquots taken from 10-fold serial dilutions of wild-type 130b (A and B) and the cas2 mutant NU411 (A) or the cas2 mutant NU412 (B) were spotted onto BCYE agar and then exposed to 0, 2.5, 5.0, 7.5, or 10 J/m² of 254-nm UV radiation. After three days of incubation, the extent of bacterial growth was determined. The results shown are representative of three independent experiments. Download

Figure S6, EPS file, 19.6 MB^{(19.6MB, eps)}

Figure S7

Sequence alignment and predicted structure of L. pneumophila Cas2. The amino acid sequence for Cas2 of strain 130b (NCBI accession no. FR687201.1) appears on the first line of text. The sequence of Cas2 from L. pneumophila Paris (NCBI accession no. NC_006368.1) is identical to that of 130b (data not shown). Below the Legionella sequence are the sequences of Cas2 proteins that have been crystallized, including ones from S. solfataricus P2 (NCBI no. AE006641.1) (45), T. thermophilus HB27 (NCBI no. AE017222.1) (45), P. furiosus DSM 3638 (NCBI no. NC_003413) (45), B. halodurans C-125 (NCBI no. Q9KFX8) (73), and D. vulgaris Hildenborough (NCBI no. NC_005863.1) (84). Residues that are conserved in all Cas2 proteins are highlighted in black, highly conserved residues are in dark gray, and similar residues are in light gray. Asterisks denote conserved residues that are important for catalytic activity (45). Secondary-structure elements derived from the predicted structure of L. pneumophila Cas2 and the crystal structures of the other Cas2 proteins are shown above the amino acid alignment. The β symbols denote four beta strand structures, and the α symbols signify three alpha-helical domains. The presence of an αβ sandwich is indicative of the ferredoxin fold (45). Download

Figure S7, EPS file, 0.9 MB^{(879.4KB, eps)}

Table S1

Primers used in this study.

Table S1, DOCX file, 0.1 MB.^{(15.9KB, docx)}

ACKNOWLEDGMENTS

We acknowledge members of the Cianciotto lab for helpful comments and advice and also thank Celeste Mallama, Richard White, and Brendan Mulhern for technical help.

F.F.G. was partly supported by NIH training grant T32 AI0007476. This work was also funded by NIH grants AI043987 and AI034937 awarded to N.P.C.

Footnotes

Citation Gunderson FF, and Cianciotto NP. 2013. The CRISPR-associated gene cas2 of Legionella pneumophila is required for intracellular infection of amoebae. mBio 4(2):e00074-13. doi:10.1128/mBio.00074-13.

REFERENCES

1. Grissa I, Vergnaud G, Pourcel C. 2007. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8:172. [DOI] [PMC free article] [PubMed] [Google Scholar]
2. Jansen R, Embden JD, Gaastra W, Schouls LM. 2002. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43:1565–1575 [DOI] [PubMed] [Google Scholar]
3. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV. 2011. Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9:467–477 [DOI] [PMC free article] [PubMed] [Google Scholar]
4. Mojica FJ, Díez-Villaseñor C, Soria E, Juez G. 2000. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol. Microbiol. 36:244–246 [DOI] [PubMed] [Google Scholar]
5. Rezzonico F, Smits TH, Duffy B. 2011. Diversity, evolution, and functionality of clustered regularly interspaced short palindromic repeat (CRISPR) regions in the fire blight pathogen Erwinia amylovora. Appl. Environ. Microbiol. 77:3819–3829 [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Touchon M, Charpentier S, Clermont O, Rocha EP, Denamur E, Branger C. 2011. CRISPR distribution within the Escherichia coli species is not suggestive of immunity-associated diversifying selection. J. Bacteriol. 193:2460–2467 [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Cady KC, White AS, Hammond JH, Abendroth MD, Karthikeyan RS, Lalitha P, Zegans ME, O’Toole GA. 2011. Prevalence, conservation and functional analysis of Yersinia and Escherichia CRISPR regions in clinical Pseudomonas aeruginosa isolates. Microbiology 157:430–437 [DOI] [PubMed] [Google Scholar]
8. Fricke WF, Mammel MK, McDermott PF, Tartera C, White DG, Leclerc JE, Ravel J, Cebula TA. 2011. Comparative genomics of 28 Salmonella enterica isolates: evidence for CRISPR-mediated adaptive sublineage evolution. J. Bacteriol. 193:3556–3568 [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Berg Miller ME, Yeoman CJ, Chia N, Tringe SG, Angly FE, Edwards RA, Flint HJ, Lamed R, Bayer EA, White BA. 2012. Phage-bacteria relationships and CRISPR elements revealed by a metagenomic survey of the rumen microbiome. Environ. Microbiol. 14:207–227 [DOI] [PubMed] [Google Scholar]
10. Garrett RA, Vestergaard G, Shah SA. 2011. Archaeal CRISPR-based immune systems: exchangeable functional modules. Trends Microbiol. 19:549–556 [DOI] [PubMed] [Google Scholar]
11. Bhaya D, Davison M, Barrangou R. 2011. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu. Rev. Genet. 45:273–297 [DOI] [PubMed] [Google Scholar]
12. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. 2008. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964 [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Lillestøl RK, Redder P, Garrett RA, Brügger K. 2006. A putative viral defence mechanism in archaeal cells. Archaea 2:59–72 [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Tang TH, Bachellerie JP, Rozhdestvensky T, Bortolin ML, Huber H, Drungowski M, Elge T, Brosius J, Hüttenhofer A. 2002. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc. Natl. Acad. Sci. U. S. A. 99:7536–7541 [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Makarova KS, Aravind L, Wolf YI, Koonin EV. 2011. Unification of cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct 6:38. [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Marraffini LA, Sontheimer EJ. 2010. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat. Rev. Genet. 11:181–190 [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Terns MP, Terns RM. 2011. CRISPR-based adaptive immune systems. Curr. Opin. Microbiol. 14:321–327 [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Haft DH, Selengut J, Mongodin EF, Nelson KE. 2005. A guild of 45 CRISPR-associated (cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol. 1:e60 http://dx.doi.org/10.1371/journal.pcbi.0010060 [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712 [DOI] [PubMed] [Google Scholar]
20. Edgar R, Qimron U. 2010. The Escherichia coli CRISPR system protects from λ lysogenization, lysogens, and prophage induction. J. Bacteriol. 192:6291–6294 [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Marraffini LA, Sontheimer EJ. 2008. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322:1843–1845 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S. 2010. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71 [DOI] [PubMed] [Google Scholar]
23. Cady KC, Bondy-Denomy J, Heussler GE, Davidson AR, O’Toole GA. 2012. The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages. J. Bacteriol. 194:5728–5738 [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Westra ER, Swarts DC, Staals RH, Jore MM, Brouns SJ, van der Oost J. 2012. The CRISPRs, they are a-changin’: how prokaryotes generate adaptive immunity. Annu. Rev. Genet. 46:311–339 [DOI] [PubMed] [Google Scholar]
25. Lopez-Sanchez MJ, Sauvage E, Da Cunha V, Clermont D, Ratsima Hariniaina E, Gonzalez-Zorn B, Poyart C, Rosinski-Chupin I, Glaser P. 2012. The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome. Mol. Microbiol. 85:1057–1071 [DOI] [PubMed] [Google Scholar]
26. Louwen R, Horst-Kreft D, de Boer AG, van der Graaf L, de Knegt G, Hamersma M, Heikema AP, Timms AR, Jacobs BC, Wagenaar JA, Endtz HP, van der Oost J, Wells JM, Nieuwenhuis EE, van Vliet AH, Willemsen PT, van Baarlen P, van Belkum A. 2013. A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome. Eur. J. Clin. Microbiol. Infect. Dis. 32:207–226 [DOI] [PubMed] [Google Scholar]
27. Nam KH, Haitjema C, Liu X, Ding F, Wang H, DeLisa MP, Ke A. 2012. Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in subtype I-C/Dvulg CRISPR-Cas system. Structure 20:1574–1584 [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Bikard D, Hatoum-Aslan A, Mucida D, Marraffini LA. 2012. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. Cell Host Microbe 12:177–186 [DOI] [PubMed] [Google Scholar]
29. Al-Attar S, Westra ER, van der Oost J, Brouns SJ. 2011. Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes. Biol. Chem. 392:277–289 [DOI] [PubMed] [Google Scholar]
30. van der Oost J, Brouns SJ. 2009. RNAi: prokaryotes get in on the act. Cell 139:863–865 [DOI] [PubMed] [Google Scholar]
31. Tyson GW, Banfield JF. 2008. Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses. Environ. Microbiol. 10:200–207 [DOI] [PubMed] [Google Scholar]
32. Hale C, Kleppe K, Terns RM, Terns MP. 2008. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 14:2572–2579 [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Lillestøl RK, Shah SA, Brügger K, Redder P, Phan H, Christiansen J, Garrett RA. 2009. CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties. Mol. Microbiol. 72:259–272 [DOI] [PubMed] [Google Scholar]
34. Semenova E, Nagornykh M, Pyatnitskiy M, Artamonova II, Severinov K. 2009. Analysis of CRISPR system function in plant pathogen Xanthomonas oryzae. FEMS Microbiol. Lett. 296:110–116 [DOI] [PubMed] [Google Scholar]
35. Pougach K, Semenova E, Bogdanova E, Datsenko KA, Djordjevic M, Wanner BL, Severinov K. 2010. Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol. Microbiol. 77:1367–1379 [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Hatoum-Aslan A, Maniv I, Marraffini LA. 2011. Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc. Natl. Acad. Sci. U. S. A. 108:21218–21222 [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Hale CR, Majumdar S, Elmore J, Pfister N, Compton M, Olson S, Resch AM, Glover CV, III, Graveley BR, Terns RM, Terns MP. 2012. Essential features and rational design of CRISPR RNAs that function with the cas RAMP module complex to cleave RNAs. Mol. Cell 45:292–302 [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA. 2010. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329:1355–1358 [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Wiedenheft B, Lander GC, Zhou K, Jore MM, Brouns SJ, van der Oost J, Doudna JA, Nogales E. 2011. Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477:486–489 [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Wiedenheft B, van Duijn E, Bultema JB, Waghmare SP, Zhou K, Barendregt A, Westphal W, Heck AJ, Boekema EJ, Dickman MJ, Doudna JA. 2011. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl. Acad. Sci. U. S. A. 108:10092–10097 [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, Severinov K. 2011. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl. Acad. Sci. U. S. A. 108:10098–10103 [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Zhang J, Rouillon C, Kerou M, Reeks J, Brugger K, Graham S, Reimann J, Cannone G, Liu H, Albers SV, Naismith JH, Spagnolo L, White MF. 2012. Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol. Cell 45:303–313 [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Wiedenheft B, Zhou K, Jinek M, Coyle SM, Ma W, Doudna JA. 2009. Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. Structure 17:904–912 [DOI] [PubMed] [Google Scholar]
44. Han D, Lehmann K, Krauss G. 2009. SSO1450—a CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA. FEBS Lett. 583:1928–1932 [DOI] [PubMed] [Google Scholar]
45. Beloglazova N, Brown G, Zimmerman MD, Proudfoot M, Makarova KS, Kudritska M, Kochinyan S, Wang S, Chruszcz M, Minor W, Koonin EV, Edwards AM, Savchenko A, Yakunin AF. 2008. A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats. J. Biol. Chem. 283:20361–20371 [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. 2006. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 1:7. [DOI] [PMC free article] [PubMed] [Google Scholar]
47. Zhang J, Kasciukovic T, White MF. 2012. The CRISPR associated protein Cas4 is a 5′ to 3′ DNA exonuclease with an iron-sulfur cluster. PLoS One 7:e47232 http://dx.doi.org/10.1371/journal.pone.0047232 [DOI] [PMC free article] [PubMed] [Google Scholar]
48. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E. 2011. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602–607 [DOI] [PMC free article] [PubMed] [Google Scholar]
49. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821 [DOI] [PMC free article] [PubMed] [Google Scholar]
50. Cady KC, O’Toole GA. 2011. Non-identity-mediated CRISPR-bacteriophage interaction mediated via the Csy and Cas3 proteins. J. Bacteriol. 193:3433–3445 [DOI] [PMC free article] [PubMed] [Google Scholar]
51. Carte J, Pfister NT, Compton MM, Terns RM, Terns MP. 2010. Binding and cleavage of CRISPR RNA by Cas6. RNA 16:2181–2188 [DOI] [PMC free article] [PubMed] [Google Scholar]
52. Zegans ME, Wagner JC, Cady KC, Murphy DM, Hammond JH, O’Toole GA. 2009. Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa. J. Bacteriol. 191:210–219 [DOI] [PMC free article] [PubMed] [Google Scholar]
53. Deng L, Kenchappa CS, Peng X, She Q, Garrett RA. 2012. Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus. Nucleic Acids Res. 40:2470–2480 [DOI] [PMC free article] [PubMed] [Google Scholar]
54. Richter H, Zoephel J, Schermuly J, Maticzka D, Backofen R, Randau L. 2012. Characterization of CRISPR RNA processing in Clostridium thermocellum and Methanococcus maripaludis. Nucleic Acids Res. 40:9887–9896 [DOI] [PMC free article] [PubMed] [Google Scholar]
55. Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E. 2005. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60:174–182 [DOI] [PubMed] [Google Scholar]
56. Takeuchi N, Wolf YI, Makarova KS, Koonin EV. 2012. Nature and intensity of selection pressure on CRISPR-associated genes. J. Bacteriol. 194:1216–1225 [DOI] [PMC free article] [PubMed] [Google Scholar]
57. Newton HJ, Ang DK, van Driel IR, Hartland EL. 2010. Molecular pathogenesis of infections caused by Legionella pneumophila. Clin. Microbiol. Rev. 23:274–298 [DOI] [PMC free article] [PubMed] [Google Scholar]
58. Fields BS, Benson RF, Besser RE. 2002. Legionella and legionnaires’ disease: 25 years of investigation. Clin. Microbiol. Rev. 15:506–526 [DOI] [PMC free article] [PubMed] [Google Scholar]
59. for Disease Control and Prevention Centers. 2011. Legionellosis—United States, 2000–2009. MMWR Morb. Mortal. Wkly. Rep. 60:1083–1086 [PubMed] [Google Scholar]
60. Valster RM, Wullings BA, van den Berg R, van der Kooij D. 2011. Relationships between free-living protozoa, cultivable Legionella spp., and water quality characteristics in three drinking water supplies in the Caribbean. Appl. Environ. Microbiol. 77:7321–7328 [DOI] [PMC free article] [PubMed] [Google Scholar]
61. Lau HY, Ashbolt NJ. 2009. The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J. Appl. Microbiol. 107:368–378 [DOI] [PubMed] [Google Scholar]
62. Taylor M, Ross K, Bentham R. 2009. Legionella, protozoa, and biofilms: interactions within complex microbial systems. Microb. Ecol. 58:538–547 [DOI] [PubMed] [Google Scholar]
63. Buse HY, Schoen ME, Ashbolt NJ. 2012. Legionellae in engineered systems and use of quantitative microbial risk assessment to predict exposure. Water Res. 46:921–933 [DOI] [PubMed] [Google Scholar]
64. Hsu BM, Huang CC, Chen JS, Chen NH, Huang JT. 2011. Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments. Water Res. 45:5171–5183 [DOI] [PubMed] [Google Scholar]
65. Swanson MS, Hammer BK. 2000. Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu. Rev. Microbiol. 54:567–613 [DOI] [PubMed] [Google Scholar]
66. Isberg RR, O’Connor TJ, Heidtman M. 2009. The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat. Rev. Microbiol. 7:13–24 [DOI] [PMC free article] [PubMed] [Google Scholar]
67. D’Auria G, Jiménez-Hernández N, Peris-Bondia F, Moya A, Latorre A. 2010. Legionella pneumophila pangenome reveals strain-specific virulence factors. BMC Genomics 11:181. [DOI] [PMC free article] [PubMed] [Google Scholar]
68. Faucher SP, Shuman HA. 2011. Small regulatory RNA and Legionella pneumophila. Front Microbiol. 2:98. [DOI] [PMC free article] [PubMed] [Google Scholar]
69. Ginevra C, Jacotin N, Diancourt L, Guigon G, Arquilliere R, Meugnier H, Descours G, Vandenesch F, Etienne J, Lina G, Caro V, Jarraud S. 2012. Legionella pneumophila sequence type 1/Paris pulsotype subtyping by spoligotyping. J. Clin. Microbiol. 50:696–701 [DOI] [PMC free article] [PubMed] [Google Scholar]
70. Grissa I, Vergnaud G, Pourcel C. 2007. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 35:W52–W57 [DOI] [PMC free article] [PubMed] [Google Scholar]
71. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. 2011. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39:9275–9282 [DOI] [PMC free article] [PubMed] [Google Scholar]
72. Molofsky AB, Swanson MS. 2004. Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol. Microbiol. 53:29–40 [DOI] [PubMed] [Google Scholar]
73. Nam KH, Ding F, Haitjema C, Huang Q, Delisa MP, Ke A. 2012. Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein. J. Biol. Chem. 287:35943–35952 [DOI] [PMC free article] [PubMed] [Google Scholar]
74. Babu M, Beloglazova N, Flick R, Graham C, Skarina T, Nocek B, Gagarinova A, Pogoutse O, Brown G, Binkowski A, Phanse S, Joachimiak A, Koonin EV, Savchenko A, Emili A, Greenblatt J, Edwards AM, Yakunin AF. 2011. A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair. Mol. Microbiol. 79:484–502 [DOI] [PMC free article] [PubMed] [Google Scholar]
75. Lammertyn E, Vande Voorde J, Meyen E, Maes L, Mast J, Anné J. 2008. Evidence for the presence of Legionella bacteriophages in environmental water samples. Microb. Ecol. 56:191–197 [DOI] [PubMed] [Google Scholar]
76. Gomez-Valero L, Rusniok C, Buchrieser C. 2009. Legionella pneumophila: population genetics, phylogeny and genomics. Infect. Genet. Evol. 9:727–739 [DOI] [PubMed] [Google Scholar]
77. Perez-Rodriguez R, Haitjema C, Huang Q, Nam KH, Bernardis S, Ke A, DeLisa MP. 2011. Envelope stress is a trigger of CRISPR RNA-mediated DNA silencing in Escherichia coli. Mol. Microbiol. 79:584–599 [DOI] [PMC free article] [PubMed] [Google Scholar]
78. Raivio T. 2011. Identifying your enemies—could envelope stress trigger microbial immunity? Mol. Microbiol. 79:557–561 [DOI] [PubMed] [Google Scholar]
79. Plagens A, Tjaden B, Hagemann A, Randau L, Hensel R. 2012. Characterization of the CRISPR/Cas subtype I-A system of the hyperthermophilic crenarchaeon Thermoproteus tenax. J. Bacteriol. 194:2491–2500 [DOI] [PMC free article] [PubMed] [Google Scholar]
80. Palmer KL, Whiteley M. 2011. DMS3-42: the secret to CRISPR-dependent biofilm inhibition in Pseudomonas aeruginosa. J. Bacteriol. 193:3431–3432 [DOI] [PMC free article] [PubMed] [Google Scholar]
81. Viswanathan P, Murphy K, Julien B, Garza AG, Kroos L. 2007. Regulation of dev, an operon that includes genes essential for Myxococcus xanthus development and CRISPR-associated genes and repeats. J. Bacteriol. 189:3738–3750 [DOI] [PMC free article] [PubMed] [Google Scholar]
82. Aklujkar M, Lovley DR. 2010. Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus. BMC Evol. Biol. 10:230. [DOI] [PMC free article] [PubMed] [Google Scholar]
83. Maris C, Dominguez C, Allain FH. 2005. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J 272:2118–2131 [DOI] [PubMed] [Google Scholar]
84. Samai P, Smith P, Shuman S. 2010. Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris. Acta Crystallogr. 66:1552–1556 [DOI] [PMC free article] [PubMed] [Google Scholar]
85. Kang SO, Caparon MG, Cho KH. 2010. Virulence gene regulation by CvfA, a putative RNase: the CvfA-enolase complex in Streptococcus pyogenes links nutritional stress, growth-phase control, and virulence gene expression. Infect. Immun. 78:2754–2767 [DOI] [PMC free article] [PubMed] [Google Scholar]
86. Schroeder GN, Petty NK, Mousnier A, Harding CR, Vogrin AJ, Wee B, Fry NK, Harrison TG, Newton HJ, Thomson NR, Beatson SA, Dougan G, Hartland EL, Frankel G. 2010. Legionella pneumophila strain 130b possesses a unique combination of type IV secretion systems and novel Dot/Icm secretion system effector proteins. J. Bacteriol. 192:6001–6016 [DOI] [PMC free article] [PubMed] [Google Scholar]
87. Brüggemann H, Hagman A, Jules M, Sismeiro O, Dillies MA, Gouyette C, Kunst F, Steinert M, Heuner K, Coppée JY, Buchrieser C. 2006. Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila. Cell. Microbiol. 8:1228–1240 [DOI] [PubMed] [Google Scholar]
88. Santic M, Ozanic M, Semic V, Pavokovic G, Mrvcic V, Kwaik YA. 2011. Intra-vacuolar proliferation of F. novicida within H. vermiformis. Front Microbiol. 2:78. [DOI] [PMC free article] [PubMed] [Google Scholar]
89. Stewart CR, Burnside DM, Cianciotto NP. 2011. The surfactant of Legionella pneumophila is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species. J. Bacteriol. 193:5971–5984 [DOI] [PMC free article] [PubMed] [Google Scholar]
90. Chatfield CH, Mulhern BJ, Burnside DM, Cianciotto NP. 2011. Legionella pneumophila LbtU acts as a novel, TonB-independent receptor for the legiobactin siderophore. J. Bacteriol. 193:1563–1575 [DOI] [PMC free article] [PubMed] [Google Scholar]
91. Bennett-Lovsey RM, Herbert AD, Sternberg MJ, Kelley LA. 2008. Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre. Proteins 70:611–625 [DOI] [PubMed] [Google Scholar]
92. Zhang Y. 2008. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40. [DOI] [PMC free article] [PubMed] [Google Scholar]
93. Koo JT, Alleyne TM, Schiano CA, Jafari N, Lathem WW. 2011. Global discovery of small RNAs in Yersinia pseudotuberculosis identifies Yersinia-specific small, noncoding RNAs required for virulence. Proc. Natl. Acad. Sci. U. S. A. 108:709–717 [DOI] [PMC free article] [PubMed] [Google Scholar]
94. Faucher SP, Mueller CA, Shuman HA. 2011. Legionella pneumophila transcriptome during intracellular multiplication in human macrophages. Front Microbiol. 2:60. [DOI] [PMC free article] [PubMed] [Google Scholar]
95. Broich M, Rydzewski K, McNealy TL, Marre R, Flieger A. 2006. The global regulatory proteins LetA and RpoS control phospholipase A, lysophospholipase A, acyltransferase, and other hydrolytic activities of Legionella pneumophila JR32. J. Bacteriol. 188:1218–1226 [DOI] [PMC free article] [PubMed] [Google Scholar]
96. Doléans-Jordheim A, Akermi M, Ginevra C, Cazalet C, Kay E, Schneider D, Buchrieser C, Atlan D, Vandenesch F, Etienne J, Jarraud S. 2006. Growth-phase-dependent mobility of the lvh-encoding region in Legionella pneumophila strain Paris. Microbiology 152:3561–3568 [DOI] [PubMed] [Google Scholar]
97. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta delta C(T)) method. Methods 25:402–408 [DOI] [PubMed] [Google Scholar]
98. McCoy-Simandle K, Stewart CR, Dao J, Debroy S, Rossier O, Bryce PJ, Cianciotto NP. 2011. Legionella pneumophila type II secretion dampens the cytokine response of infected macrophages and epithelia. Infect. Immun. 79:1984–1997 [DOI] [PMC free article] [PubMed] [Google Scholar]
99. Charpentier X, Kay E, Schneider D, Shuman HA. 2011. Antibiotics and UV radiation induce competence for natural transformation in Legionella pneumophila. J. Bacteriol. 193:1114–1121 [DOI] [PMC free article] [PubMed] [Google Scholar]
100. Knudson GB. 1985. Photoreactivation of UV-irradiated Legionella pneumophila and other Legionella species. Appl. Environ. Microbiol. 49:975–980 [DOI] [PMC free article] [PubMed] [Google Scholar]
101. Courcelle J, Crowley DJ, Hanawalt PC. 1999. Recovery of DNA replication in UV-irradiated Escherichia coli requires both excision repair and recF protein function. J. Bacteriol. 181:916–922 [DOI] [PMC free article] [PubMed] [Google Scholar]
102. Al-Deib AA, Mahdi AA, Lloyd RG. 1996. Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12. J. Bacteriol. 178:6782–6789 [DOI] [PMC free article] [PubMed] [Google Scholar]
103. Yoshida S, Mizuguchi Y. 1984. Antibiotic susceptibility of Legionella pneumophia Philadelphia-1 in cultured guinea-pig peritoneal macrophages. J. Gen. Microbiol. 130:901–906 [DOI] [PubMed] [Google Scholar]
104. Robey M, O’Connell W, Cianciotto NP. 2001. Identification of Legionella pneumophila rcp, a pagP-like gene that confers resistance to cationic antimicrobial peptides and promotes intracellular infection. Infect. Immun. 69:4276–4286 [DOI] [PMC free article] [PubMed] [Google Scholar]
105. Hazan R, Sat B, Engelberg-Kulka H. 2004. Escherichia coli mazEF-mediated cell death is triggered by various stressful conditions. J. Bacteriol. 186:3663–3669 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1

Figure S1, EPS file, 2.7 MB^{(2.7MB, eps)}

Figure S2

Figure S2, EPS file, 2.2 MB^{(2.2MB, eps)}

Figure S3

Figure S3, EPS file, 0.4 MB^{(453.4KB, eps)}

Figure S4

Figure S4, EPS file, 19.6 MB^{(19.6MB, eps)}

Figure S5

Figure S5, EPS file, 0.5 MB^{(503.9KB, eps)}

Figure S6

Figure S6, EPS file, 19.6 MB^{(19.6MB, eps)}

Figure S7

Figure S7, EPS file, 0.9 MB^{(879.4KB, eps)}

Table S1

Primers used in this study.

Table S1, DOCX file, 0.1 MB.^{(15.9KB, docx)}

[B1] 1. Grissa I, Vergnaud G, Pourcel C. 2007. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8:172. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2. Jansen R, Embden JD, Gaastra W, Schouls LM. 2002. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43:1565–1575 [DOI] [PubMed] [Google Scholar]

[B3] 3. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV. 2011. Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9:467–477 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Mojica FJ, Díez-Villaseñor C, Soria E, Juez G. 2000. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol. Microbiol. 36:244–246 [DOI] [PubMed] [Google Scholar]

[B5] 5. Rezzonico F, Smits TH, Duffy B. 2011. Diversity, evolution, and functionality of clustered regularly interspaced short palindromic repeat (CRISPR) regions in the fire blight pathogen Erwinia amylovora. Appl. Environ. Microbiol. 77:3819–3829 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Touchon M, Charpentier S, Clermont O, Rocha EP, Denamur E, Branger C. 2011. CRISPR distribution within the Escherichia coli species is not suggestive of immunity-associated diversifying selection. J. Bacteriol. 193:2460–2467 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7. Cady KC, White AS, Hammond JH, Abendroth MD, Karthikeyan RS, Lalitha P, Zegans ME, O’Toole GA. 2011. Prevalence, conservation and functional analysis of Yersinia and Escherichia CRISPR regions in clinical Pseudomonas aeruginosa isolates. Microbiology 157:430–437 [DOI] [PubMed] [Google Scholar]

[B8] 8. Fricke WF, Mammel MK, McDermott PF, Tartera C, White DG, Leclerc JE, Ravel J, Cebula TA. 2011. Comparative genomics of 28 Salmonella enterica isolates: evidence for CRISPR-mediated adaptive sublineage evolution. J. Bacteriol. 193:3556–3568 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9. Berg Miller ME, Yeoman CJ, Chia N, Tringe SG, Angly FE, Edwards RA, Flint HJ, Lamed R, Bayer EA, White BA. 2012. Phage-bacteria relationships and CRISPR elements revealed by a metagenomic survey of the rumen microbiome. Environ. Microbiol. 14:207–227 [DOI] [PubMed] [Google Scholar]

[B10] 10. Garrett RA, Vestergaard G, Shah SA. 2011. Archaeal CRISPR-based immune systems: exchangeable functional modules. Trends Microbiol. 19:549–556 [DOI] [PubMed] [Google Scholar]

[B11] 11. Bhaya D, Davison M, Barrangou R. 2011. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu. Rev. Genet. 45:273–297 [DOI] [PubMed] [Google Scholar]

[B12] 12. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. 2008. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Lillestøl RK, Redder P, Garrett RA, Brügger K. 2006. A putative viral defence mechanism in archaeal cells. Archaea 2:59–72 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14. Tang TH, Bachellerie JP, Rozhdestvensky T, Bortolin ML, Huber H, Drungowski M, Elge T, Brosius J, Hüttenhofer A. 2002. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc. Natl. Acad. Sci. U. S. A. 99:7536–7541 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15. Makarova KS, Aravind L, Wolf YI, Koonin EV. 2011. Unification of cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct 6:38. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. Marraffini LA, Sontheimer EJ. 2010. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat. Rev. Genet. 11:181–190 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17. Terns MP, Terns RM. 2011. CRISPR-based adaptive immune systems. Curr. Opin. Microbiol. 14:321–327 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18. Haft DH, Selengut J, Mongodin EF, Nelson KE. 2005. A guild of 45 CRISPR-associated (cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol. 1:e60 http://dx.doi.org/10.1371/journal.pcbi.0010060 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712 [DOI] [PubMed] [Google Scholar]

[B20] 20. Edgar R, Qimron U. 2010. The Escherichia coli CRISPR system protects from λ lysogenization, lysogens, and prophage induction. J. Bacteriol. 192:6291–6294 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21. Marraffini LA, Sontheimer EJ. 2008. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322:1843–1845 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S. 2010. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71 [DOI] [PubMed] [Google Scholar]

[B23] 23. Cady KC, Bondy-Denomy J, Heussler GE, Davidson AR, O’Toole GA. 2012. The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages. J. Bacteriol. 194:5728–5738 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24. Westra ER, Swarts DC, Staals RH, Jore MM, Brouns SJ, van der Oost J. 2012. The CRISPRs, they are a-changin’: how prokaryotes generate adaptive immunity. Annu. Rev. Genet. 46:311–339 [DOI] [PubMed] [Google Scholar]

[B25] 25. Lopez-Sanchez MJ, Sauvage E, Da Cunha V, Clermont D, Ratsima Hariniaina E, Gonzalez-Zorn B, Poyart C, Rosinski-Chupin I, Glaser P. 2012. The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome. Mol. Microbiol. 85:1057–1071 [DOI] [PubMed] [Google Scholar]

[B26] 26. Louwen R, Horst-Kreft D, de Boer AG, van der Graaf L, de Knegt G, Hamersma M, Heikema AP, Timms AR, Jacobs BC, Wagenaar JA, Endtz HP, van der Oost J, Wells JM, Nieuwenhuis EE, van Vliet AH, Willemsen PT, van Baarlen P, van Belkum A. 2013. A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome. Eur. J. Clin. Microbiol. Infect. Dis. 32:207–226 [DOI] [PubMed] [Google Scholar]

[B27] 27. Nam KH, Haitjema C, Liu X, Ding F, Wang H, DeLisa MP, Ke A. 2012. Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in subtype I-C/Dvulg CRISPR-Cas system. Structure 20:1574–1584 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28. Bikard D, Hatoum-Aslan A, Mucida D, Marraffini LA. 2012. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. Cell Host Microbe 12:177–186 [DOI] [PubMed] [Google Scholar]

[B29] 29. Al-Attar S, Westra ER, van der Oost J, Brouns SJ. 2011. Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes. Biol. Chem. 392:277–289 [DOI] [PubMed] [Google Scholar]

[B30] 30. van der Oost J, Brouns SJ. 2009. RNAi: prokaryotes get in on the act. Cell 139:863–865 [DOI] [PubMed] [Google Scholar]

[B31] 31. Tyson GW, Banfield JF. 2008. Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses. Environ. Microbiol. 10:200–207 [DOI] [PubMed] [Google Scholar]

[B32] 32. Hale C, Kleppe K, Terns RM, Terns MP. 2008. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 14:2572–2579 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Lillestøl RK, Shah SA, Brügger K, Redder P, Phan H, Christiansen J, Garrett RA. 2009. CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties. Mol. Microbiol. 72:259–272 [DOI] [PubMed] [Google Scholar]

[B34] 34. Semenova E, Nagornykh M, Pyatnitskiy M, Artamonova II, Severinov K. 2009. Analysis of CRISPR system function in plant pathogen Xanthomonas oryzae. FEMS Microbiol. Lett. 296:110–116 [DOI] [PubMed] [Google Scholar]

[B35] 35. Pougach K, Semenova E, Bogdanova E, Datsenko KA, Djordjevic M, Wanner BL, Severinov K. 2010. Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol. Microbiol. 77:1367–1379 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36. Hatoum-Aslan A, Maniv I, Marraffini LA. 2011. Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc. Natl. Acad. Sci. U. S. A. 108:21218–21222 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37. Hale CR, Majumdar S, Elmore J, Pfister N, Compton M, Olson S, Resch AM, Glover CV, III, Graveley BR, Terns RM, Terns MP. 2012. Essential features and rational design of CRISPR RNAs that function with the cas RAMP module complex to cleave RNAs. Mol. Cell 45:292–302 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38. Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA. 2010. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329:1355–1358 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39. Wiedenheft B, Lander GC, Zhou K, Jore MM, Brouns SJ, van der Oost J, Doudna JA, Nogales E. 2011. Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477:486–489 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40. Wiedenheft B, van Duijn E, Bultema JB, Waghmare SP, Zhou K, Barendregt A, Westphal W, Heck AJ, Boekema EJ, Dickman MJ, Doudna JA. 2011. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl. Acad. Sci. U. S. A. 108:10092–10097 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B41] 41. Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, Severinov K. 2011. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl. Acad. Sci. U. S. A. 108:10098–10103 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42. Zhang J, Rouillon C, Kerou M, Reeks J, Brugger K, Graham S, Reimann J, Cannone G, Liu H, Albers SV, Naismith JH, Spagnolo L, White MF. 2012. Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol. Cell 45:303–313 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43. Wiedenheft B, Zhou K, Jinek M, Coyle SM, Ma W, Doudna JA. 2009. Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. Structure 17:904–912 [DOI] [PubMed] [Google Scholar]

[B44] 44. Han D, Lehmann K, Krauss G. 2009. SSO1450—a CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA. FEBS Lett. 583:1928–1932 [DOI] [PubMed] [Google Scholar]

[B45] 45. Beloglazova N, Brown G, Zimmerman MD, Proudfoot M, Makarova KS, Kudritska M, Kochinyan S, Wang S, Chruszcz M, Minor W, Koonin EV, Edwards AM, Savchenko A, Yakunin AF. 2008. A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats. J. Biol. Chem. 283:20361–20371 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B46] 46. Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. 2006. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 1:7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B47] 47. Zhang J, Kasciukovic T, White MF. 2012. The CRISPR associated protein Cas4 is a 5′ to 3′ DNA exonuclease with an iron-sulfur cluster. PLoS One 7:e47232 http://dx.doi.org/10.1371/journal.pone.0047232 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B48] 48. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E. 2011. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602–607 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B49] 49. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B50] 50. Cady KC, O’Toole GA. 2011. Non-identity-mediated CRISPR-bacteriophage interaction mediated via the Csy and Cas3 proteins. J. Bacteriol. 193:3433–3445 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B51] 51. Carte J, Pfister NT, Compton MM, Terns RM, Terns MP. 2010. Binding and cleavage of CRISPR RNA by Cas6. RNA 16:2181–2188 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B52] 52. Zegans ME, Wagner JC, Cady KC, Murphy DM, Hammond JH, O’Toole GA. 2009. Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa. J. Bacteriol. 191:210–219 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B53] 53. Deng L, Kenchappa CS, Peng X, She Q, Garrett RA. 2012. Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus. Nucleic Acids Res. 40:2470–2480 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B54] 54. Richter H, Zoephel J, Schermuly J, Maticzka D, Backofen R, Randau L. 2012. Characterization of CRISPR RNA processing in Clostridium thermocellum and Methanococcus maripaludis. Nucleic Acids Res. 40:9887–9896 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B55] 55. Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E. 2005. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60:174–182 [DOI] [PubMed] [Google Scholar]

[B56] 56. Takeuchi N, Wolf YI, Makarova KS, Koonin EV. 2012. Nature and intensity of selection pressure on CRISPR-associated genes. J. Bacteriol. 194:1216–1225 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B57] 57. Newton HJ, Ang DK, van Driel IR, Hartland EL. 2010. Molecular pathogenesis of infections caused by Legionella pneumophila. Clin. Microbiol. Rev. 23:274–298 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B58] 58. Fields BS, Benson RF, Besser RE. 2002. Legionella and legionnaires’ disease: 25 years of investigation. Clin. Microbiol. Rev. 15:506–526 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B59] 59. for Disease Control and Prevention Centers. 2011. Legionellosis—United States, 2000–2009. MMWR Morb. Mortal. Wkly. Rep. 60:1083–1086 [PubMed] [Google Scholar]

[B60] 60. Valster RM, Wullings BA, van den Berg R, van der Kooij D. 2011. Relationships between free-living protozoa, cultivable Legionella spp., and water quality characteristics in three drinking water supplies in the Caribbean. Appl. Environ. Microbiol. 77:7321–7328 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B61] 61. Lau HY, Ashbolt NJ. 2009. The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J. Appl. Microbiol. 107:368–378 [DOI] [PubMed] [Google Scholar]

[B62] 62. Taylor M, Ross K, Bentham R. 2009. Legionella, protozoa, and biofilms: interactions within complex microbial systems. Microb. Ecol. 58:538–547 [DOI] [PubMed] [Google Scholar]

[B63] 63. Buse HY, Schoen ME, Ashbolt NJ. 2012. Legionellae in engineered systems and use of quantitative microbial risk assessment to predict exposure. Water Res. 46:921–933 [DOI] [PubMed] [Google Scholar]

[B64] 64. Hsu BM, Huang CC, Chen JS, Chen NH, Huang JT. 2011. Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments. Water Res. 45:5171–5183 [DOI] [PubMed] [Google Scholar]

[B65] 65. Swanson MS, Hammer BK. 2000. Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu. Rev. Microbiol. 54:567–613 [DOI] [PubMed] [Google Scholar]

[B66] 66. Isberg RR, O’Connor TJ, Heidtman M. 2009. The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat. Rev. Microbiol. 7:13–24 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B67] 67. D’Auria G, Jiménez-Hernández N, Peris-Bondia F, Moya A, Latorre A. 2010. Legionella pneumophila pangenome reveals strain-specific virulence factors. BMC Genomics 11:181. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B68] 68. Faucher SP, Shuman HA. 2011. Small regulatory RNA and Legionella pneumophila. Front Microbiol. 2:98. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B69] 69. Ginevra C, Jacotin N, Diancourt L, Guigon G, Arquilliere R, Meugnier H, Descours G, Vandenesch F, Etienne J, Lina G, Caro V, Jarraud S. 2012. Legionella pneumophila sequence type 1/Paris pulsotype subtyping by spoligotyping. J. Clin. Microbiol. 50:696–701 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B70] 70. Grissa I, Vergnaud G, Pourcel C. 2007. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 35:W52–W57 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B71] 71. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. 2011. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39:9275–9282 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B72] 72. Molofsky AB, Swanson MS. 2004. Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol. Microbiol. 53:29–40 [DOI] [PubMed] [Google Scholar]

[B73] 73. Nam KH, Ding F, Haitjema C, Huang Q, Delisa MP, Ke A. 2012. Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein. J. Biol. Chem. 287:35943–35952 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B74] 74. Babu M, Beloglazova N, Flick R, Graham C, Skarina T, Nocek B, Gagarinova A, Pogoutse O, Brown G, Binkowski A, Phanse S, Joachimiak A, Koonin EV, Savchenko A, Emili A, Greenblatt J, Edwards AM, Yakunin AF. 2011. A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair. Mol. Microbiol. 79:484–502 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B75] 75. Lammertyn E, Vande Voorde J, Meyen E, Maes L, Mast J, Anné J. 2008. Evidence for the presence of Legionella bacteriophages in environmental water samples. Microb. Ecol. 56:191–197 [DOI] [PubMed] [Google Scholar]

[B76] 76. Gomez-Valero L, Rusniok C, Buchrieser C. 2009. Legionella pneumophila: population genetics, phylogeny and genomics. Infect. Genet. Evol. 9:727–739 [DOI] [PubMed] [Google Scholar]

[B77] 77. Perez-Rodriguez R, Haitjema C, Huang Q, Nam KH, Bernardis S, Ke A, DeLisa MP. 2011. Envelope stress is a trigger of CRISPR RNA-mediated DNA silencing in Escherichia coli. Mol. Microbiol. 79:584–599 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B78] 78. Raivio T. 2011. Identifying your enemies—could envelope stress trigger microbial immunity? Mol. Microbiol. 79:557–561 [DOI] [PubMed] [Google Scholar]

[B79] 79. Plagens A, Tjaden B, Hagemann A, Randau L, Hensel R. 2012. Characterization of the CRISPR/Cas subtype I-A system of the hyperthermophilic crenarchaeon Thermoproteus tenax. J. Bacteriol. 194:2491–2500 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B80] 80. Palmer KL, Whiteley M. 2011. DMS3-42: the secret to CRISPR-dependent biofilm inhibition in Pseudomonas aeruginosa. J. Bacteriol. 193:3431–3432 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B81] 81. Viswanathan P, Murphy K, Julien B, Garza AG, Kroos L. 2007. Regulation of dev, an operon that includes genes essential for Myxococcus xanthus development and CRISPR-associated genes and repeats. J. Bacteriol. 189:3738–3750 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B82] 82. Aklujkar M, Lovley DR. 2010. Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus. BMC Evol. Biol. 10:230. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B83] 83. Maris C, Dominguez C, Allain FH. 2005. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J 272:2118–2131 [DOI] [PubMed] [Google Scholar]

[B84] 84. Samai P, Smith P, Shuman S. 2010. Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris. Acta Crystallogr. 66:1552–1556 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B85] 85. Kang SO, Caparon MG, Cho KH. 2010. Virulence gene regulation by CvfA, a putative RNase: the CvfA-enolase complex in Streptococcus pyogenes links nutritional stress, growth-phase control, and virulence gene expression. Infect. Immun. 78:2754–2767 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B86] 86. Schroeder GN, Petty NK, Mousnier A, Harding CR, Vogrin AJ, Wee B, Fry NK, Harrison TG, Newton HJ, Thomson NR, Beatson SA, Dougan G, Hartland EL, Frankel G. 2010. Legionella pneumophila strain 130b possesses a unique combination of type IV secretion systems and novel Dot/Icm secretion system effector proteins. J. Bacteriol. 192:6001–6016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B87] 87. Brüggemann H, Hagman A, Jules M, Sismeiro O, Dillies MA, Gouyette C, Kunst F, Steinert M, Heuner K, Coppée JY, Buchrieser C. 2006. Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila. Cell. Microbiol. 8:1228–1240 [DOI] [PubMed] [Google Scholar]

[B88] 88. Santic M, Ozanic M, Semic V, Pavokovic G, Mrvcic V, Kwaik YA. 2011. Intra-vacuolar proliferation of F. novicida within H. vermiformis. Front Microbiol. 2:78. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B89] 89. Stewart CR, Burnside DM, Cianciotto NP. 2011. The surfactant of Legionella pneumophila is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species. J. Bacteriol. 193:5971–5984 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B90] 90. Chatfield CH, Mulhern BJ, Burnside DM, Cianciotto NP. 2011. Legionella pneumophila LbtU acts as a novel, TonB-independent receptor for the legiobactin siderophore. J. Bacteriol. 193:1563–1575 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B91] 91. Bennett-Lovsey RM, Herbert AD, Sternberg MJ, Kelley LA. 2008. Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre. Proteins 70:611–625 [DOI] [PubMed] [Google Scholar]

[B92] 92. Zhang Y. 2008. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B93] 93. Koo JT, Alleyne TM, Schiano CA, Jafari N, Lathem WW. 2011. Global discovery of small RNAs in Yersinia pseudotuberculosis identifies Yersinia-specific small, noncoding RNAs required for virulence. Proc. Natl. Acad. Sci. U. S. A. 108:709–717 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B94] 94. Faucher SP, Mueller CA, Shuman HA. 2011. Legionella pneumophila transcriptome during intracellular multiplication in human macrophages. Front Microbiol. 2:60. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B95] 95. Broich M, Rydzewski K, McNealy TL, Marre R, Flieger A. 2006. The global regulatory proteins LetA and RpoS control phospholipase A, lysophospholipase A, acyltransferase, and other hydrolytic activities of Legionella pneumophila JR32. J. Bacteriol. 188:1218–1226 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B96] 96. Doléans-Jordheim A, Akermi M, Ginevra C, Cazalet C, Kay E, Schneider D, Buchrieser C, Atlan D, Vandenesch F, Etienne J, Jarraud S. 2006. Growth-phase-dependent mobility of the lvh-encoding region in Legionella pneumophila strain Paris. Microbiology 152:3561–3568 [DOI] [PubMed] [Google Scholar]

[B97] 97. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta delta C(T)) method. Methods 25:402–408 [DOI] [PubMed] [Google Scholar]

[B98] 98. McCoy-Simandle K, Stewart CR, Dao J, Debroy S, Rossier O, Bryce PJ, Cianciotto NP. 2011. Legionella pneumophila type II secretion dampens the cytokine response of infected macrophages and epithelia. Infect. Immun. 79:1984–1997 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B99] 99. Charpentier X, Kay E, Schneider D, Shuman HA. 2011. Antibiotics and UV radiation induce competence for natural transformation in Legionella pneumophila. J. Bacteriol. 193:1114–1121 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B100] 100. Knudson GB. 1985. Photoreactivation of UV-irradiated Legionella pneumophila and other Legionella species. Appl. Environ. Microbiol. 49:975–980 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B101] 101. Courcelle J, Crowley DJ, Hanawalt PC. 1999. Recovery of DNA replication in UV-irradiated Escherichia coli requires both excision repair and recF protein function. J. Bacteriol. 181:916–922 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B102] 102. Al-Deib AA, Mahdi AA, Lloyd RG. 1996. Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12. J. Bacteriol. 178:6782–6789 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B103] 103. Yoshida S, Mizuguchi Y. 1984. Antibiotic susceptibility of Legionella pneumophia Philadelphia-1 in cultured guinea-pig peritoneal macrophages. J. Gen. Microbiol. 130:901–906 [DOI] [PubMed] [Google Scholar]

[B104] 104. Robey M, O’Connell W, Cianciotto NP. 2001. Identification of Legionella pneumophila rcp, a pagP-like gene that confers resistance to cationic antimicrobial peptides and promotes intracellular infection. Infect. Immun. 69:4276–4286 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B105] 105. Hazan R, Sat B, Engelberg-Kulka H. 2004. Escherichia coli mazEF-mediated cell death is triggered by various stressful conditions. J. Bacteriol. 186:3663–3669 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

The CRISPR-Associated Gene cas2 of Legionella pneumophila Is Required for Intracellular Infection of Amoebae

Felizza F Gunderson

Nicholas P Cianciotto

ABSTRACT

IMPORTANCE

Introduction

RESULTS

Detection of a CRISPR-Cas locus in L. pneumophila strain 130b.

FIG 1.

Expression of the CRISPR-Cas locus in L. pneumophila grown extracellularly.

FIG 2.

Expression of the CRISPR-Cas locus in L. pneumophila grown intracellularly.

FIG 3.

FIG 4.

Isolation of L. pneumophila CRISPR-Cas mutants.

Intracellular infection by L. pneumophila CRISPR-Cas mutants.

FIG 5.

FIG 6.

DISCUSSION

MATERIALS AND METHODS

Bacterial strains, media, and extracellular growth assessments.

TABLE 1.

DNA and protein sequence analysis.

RT-PCR analysis.

Quantitative RT-PCR.

Mutant construction and genetic complementation.

Intracellular infection assays.

Assays for bacterial sensitivity to DNA-damage.

SUPPLEMENTAL MATERIAL

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

Cite

Add to Collections

PERMALINK

The CRISPR-Associated Gene cas2 of Legionella pneumophila Is Required for Intracellular Infection of Amoebae

Felizza F Gunderson

Nicholas P Cianciotto

ABSTRACT

IMPORTANCE

Introduction

RESULTS

Detection of a CRISPR-Cas locus in L. pneumophila strain 130b.

FIG 1.

Expression of the CRISPR-Cas locus in L. pneumophila grown extracellularly.

FIG 2.

Expression of the CRISPR-Cas locus in L. pneumophila grown intracellularly.

FIG 3.

FIG 4.

Isolation of L. pneumophila CRISPR-Cas mutants.

Intracellular infection by L. pneumophila CRISPR-Cas mutants.

FIG 5.

FIG 6.

DISCUSSION

MATERIALS AND METHODS

Bacterial strains, media, and extracellular growth assessments.

TABLE 1.

DNA and protein sequence analysis.

RT-PCR analysis.

Quantitative RT-PCR.

Mutant construction and genetic complementation.

Intracellular infection assays.

Assays for bacterial sensitivity to DNA-damage.

SUPPLEMENTAL MATERIAL

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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