A Novel Legionella Genomic Island Encodes a Copper-Responsive Regulatory System and a Single Icm/Dot Effector Protein Transcriptionally Activated by Copper.

Legionella pneumophila is an intracellular human pathogen that utilizes amoebae as its environmental host. The adaptation of L. pneumophila to the intracellular environment requires coordination of expression of its multicomponent pathogenesis system, which is composed of a secretion system and effector proteins. However, the regulatory factors controlling the expression of this pathogenesis system are only partially uncovered. Here, we discovered a novel regulatory system that is activated by copper and controls the expression of a single effector protein. The genes encoding both the regulatory system and the effector protein are located on a genomic island that undergoes horizontal gene transfer within the Legionella genus. This regulator-effector genomic island represents the first reported case of local regulation of effectors in Legionella. The discovery of this regulatory mechanism is an important step forward in the understanding of how the regulatory network of effectors functions and evolves in the Legionella genus.

L egionella pneumophila is an intracellular human pathogen that multiplies within alveolar macrophages and causes a severe pneumonia known as Legionnaires' disease (1)(2)(3). In the environment, L. pneumophila thrives in many different protozoan cells (4)(5)(6), which serve as its training grounds for pathogenesis (7). Inside its eukaryotic host cells, the bacterium remodels its phagosome to generate the Legionella-containing vacuole (LCV) (8,9). Establishment of the LCV depends on the Icm/Dot type IV secretion system, which delivers more than 300 effector proteins, which modulate numerous host-cell functions during infection (10)(11)(12)(13)(14). The enormous number of effectors that participate in LCV establishment and the various host cell pathways manipulated by L. pneumophila effectors (15)(16)(17) imply that a successful infection will require different levels of coordination among the effectors, including on the level of gene expression.
To date, five regulatory systems have been shown to directly regulate the expression of effector-encoding genes (EEGs) in L. pneumophila: (i) the PmrAB two-component system (TCS) activates the expression of about 40 EEGs (18,19); (ii) the CpxRA TCS activates or represses the expression of about 30 EEGs and also regulates the expression of several icm/dot genes (20)(21)(22)(23); (iii) the LetAS-RsmYZ-CsrA regulatory cascade represses the expression of about 40 EEGs during exponential phase (24)(25)(26)(27)(28)(29)(30)(31); (iv) two Fis regulators repress the expression of about 20 EEGs during exponential phase (32); and (v) the Fur regulator controls the expression of a single EEG (mavN) as well as several other proteins involved in iron acquisition (33)(34)(35)(36)(37). In addition, these regulatory systems have been shown to assemble into an interconnected regulatory network using accessory components, such as modulators (AckA-Pta and PTS Ntr [22,38]) and connectors (LetE and LerC [22,39,40]). All the direct regulators of EEGs described above (PmrA, CpxR, CsrA, Fis1, Fis3, and Fur) were found to be present in all the Legionella species examined (41,42), and they function as global regulators that regulate the expression of a large number of genes, including EEGs, scattered throughout the L. pneumophila genome (18)(19)(20)22). Besides global regulators, local regulators that regulate the expression of a small number of adjacent genes are also common in many bacterial systems (43,44), including pathogenesis-related genes in Vibrio cholerae and Salmonella enterica (45). Local regulators were shown to function as either activators or repressors, and in many cases, they are found in genomic islands together with the genes they regulate (46)(47)(48)(49).
Genomic islands are genetic elements acquired via horizontal gene transfer (HGT) that include sets of genes that encode proteins that may be beneficial for the bacteria under certain conditions (50,51). Many pathogenicity islands have been described in bacteria, some of which contain a large number of genes and encode complete pathogenesis systems (50,52). For example, the Salmonella enterica pathogenicity island 2 (SPI-2) encodes the complex components of a type III system, effector proteins, and the SsrAB TCS, which functions as a local regulator and coordinates their expression (53). Smaller pathogenicity islands that encode a few proteins are sometimes referred to as islets (50). For example, the Streptococcus pneumoniae RlrA pathogenicity islet encodes the RlrA local transcriptional regulator, which controls the expression of genes located on the same islet that are essential for lung infection (54). Another example is the Listeria monocytogenes virulence gene cluster that encodes phospholipases, listeriolysin, metalloprotease, the ActA protein, and the PrfA transcriptional regulator, which controls their expression as well as the expression of genes outside the island (55).
All known L. pneumophila TCSs that regulate the expression of EEGs are global regulators found in all the Legionella species sequenced. They are not part of genomic islands, and the signal sensed by their cognate sensor histidine kinases (SHKs) is unknown. Here, we describe a novel L. pneumophila TCS effector island. The TCS is composed of the LciS SHK, which specifically senses copper and activates the cognate LciR response regulator (RR). LciR functions as a local regulator, activating the expression of a single adjacent EEG (lciE). The LciRS-LciE genomic island undergoes HGT throughout the Legionella genus and represents a novel type of effector regulation in Legionella.

RESULTS
The CpxR and PmrA direct regulators of effector-encoding genes (EEGs) belong to the winged helix-turn-helix (wHTH) family of response regulators (RRs), which function as part of the CpxRA and PmrAB two-component systems (TCSs) (18-20, 22, 23). L. pneumophila harbors a third TCS from the same family, consisting of lpg0714, which encodes a sensor-histidine kinase (SHK), and lpg0715, which encodes a wHTH-type RR. This TCS is found in L. pneumophila, in five other characterized Legionella species, and in three uncharacterized Legionella species. In all these species, a gene, which was shown in L. pneumophila to encode an effector protein (56) (lpg0716 in L. pneumophila), is located next to it, forming a regulator-effector island ( Fig. 1A; see also Fig. S1 in the supplemental material).
The lpg0714-lpg0715-lpg0716 island undergoes horizontal gene transfer in the Legionella genus. Comparison of the genomic location of the regulator-effector island in the nine Legionella species in which it was found indicated that it is usually located in different positions (Fig. 1A). However, in genomes of the four closely related species (L. worsleiensis, L. moravica, L. quateirensis, and Legionella sp. strain Km535), it is positioned in a similar genomic region, which underwent additional changes in its gene content (Fig. 1A). Comparison of the genomic region of this regulator-effector island is found in L. pneumophila and in the other eight Legionella species revealed that it contains highly variable genes (Fig. S2A), indicating that it is prone to changes in gene content. To examine whether this regulator-effector island undergoes HGT in the Legionella genus, we reconstructed the phylogenetic tree of these nine species based on the protein sequences encoded by the lpg0714-lpg0715 TCS and the lpg0716 effector and compared it to the phylogenetic tree reconstructed using a TCS (PmrAB) and a core effector (LegA3) present in all the Legionella species. While the analysis using PmrAB-LegA3 resulted in a tree structure similar to that of the known Legionella phylogenetic tree (as far as the characterized Legionella species are concerned [41]), the tree based on the regulator-effector island resulted in a different topology (Fig. 1B). Moreover, while the GC content of the pmrAB-legA3 DNA sequence was similar to the genomic GC content in all nine species (Fig. S2B), the GC content of the regulatoreffector island in three of the species (L. pneumophila, L. brunensis, and L. nautarum) was considerably lower, suggesting a recent event of HGT in these species. The genomic position, the GC content, and the tree structure of L. worsleiensis, L. moravica, L. quateirensis, and Legionella sp. strain Km535 imply a single HGT event, which occurred before their speciation. Collectively, these results suggest that this regulator-effector island undergoes HGT in the Legionella genus as a unit.
The L. pneumophila lpg0714-lpg0715 TCS is homologous to TCSs involved in copper sensing in other bacteria. Examination of the proteins encoded by lpg0714 and lpg0715 indicated that homologous TCSs are present in many bacteria. In most of them, the genes located next to it encode different proteins and systems involved in copper resistance (Fig. 2); however, cases of genes encoding resistance to silver and zinc were also described (57,58). A few of these systems were studied before, including the CusRS TCS of Escherichia coli, which is located next to the CusCFBA copper transport system (59,60). Examination of the lpg0716 EEG and of all other genes located next to the homologous TCSs from the different bacteria indicated that a conserved sequence is found at a precise distance from the predicted or validated Ϫ10 promoter elements in one or two of the surrounding genes ( Fig. 2 and Fig. S3).
Due to the homology of this TCS and its predicted regulatory element to systems involved in copper sensing and detoxification, we named the L. pneumophila lpg0714-lpg0716 genes lci, for Legionella copper island. The SHK lpg0714 was named LciS, the RR lpg0715 was named LciR, and the effector lpg0716 was named LciE. lciE is induced by copper in L. pneumophila in an LciRS-dependent manner. To determine whether lciE is induced in response to copper exposure, two fusions were constructed (Fig. 3A). The first fusion contains the 300-bp regulatory region of lciE fused to lacZ (designated lciE-lacZ). The second construct contains the same fusion as well as the lciRS genes in their original genomic organization (designated lciRS-lciE-lacZ) (Fig. 3A). To examine the expression of these constructs in response to copper exposure, we first determined the concentrations of copper that L. pneumophila can tolerate (Fig. S4A) and examined the effect of copper on the expression of lciE using a range of concentrations, from 1 to 50 M. Using the lciE-lacZ fusion, the level of expression of lciE increased gradually as the concentration of copper was elevated, reaching more than 100-fold induction at the highest copper concentration (Fig. 3B). To determine the importance of the LciRS TCS for LciE expression, four deletion mutants were constructed, lciR, lciS, and lciE single deletion mutants and an lciRS-lciE triple deletion mutant. None of these mutants had an intracellular growth phenotype when examined in the amoeba host Acanthamoeba castellanii and in HL-60-derived human macrophages, as well as in a competition assay in amoeba with and without the addition of copper (Fig. S5). The copper induction of lciE was found to be completely dependent on the lciR and lciS genes, and as expected, the lciE deletion did not affect the levels of expression of the lciE-lacZ fusion (Fig. 3B). To further substantiate these results, copper induction was examined in an L. pneumophila strain deleted for the entire lciRS-lciE region (Fig. 3C). Using the lciE-lacZ fusion (Fig. 3A), no induction by copper was obtained, but when the lciRS-lciE-lacZ fusion (Fig. 3A) was examined, high levels of expression were obtained due to exposure to copper, which increased gradually as the concentrations of copper increased (Fig. 3C). In addition, the expression of lciE was found to be completely dependent on the presence of a functional LciRS TCS, since no induction was obtained when the conserved histidine of the LciS SHK and conserved aspartic acid of the LciR RR were mutated (Fig. S6A). Collectively, these results indicate that the expression of the Icm/Dot effector LciE is activated by copper, and its activation is completely dependent on the presence and functionality of the LciRS TCS. lciE is induced in E. coli after copper exposure in a CusRS-or LciRS-dependent manner. As indicated above, the LciRS homologous TCSs as well as the LciR predicted regulatory element are conserved in many bacteria. To examine if these TCSs function similarly, we examined the expression of the lciE-lacZ construct (Fig. 3A) in E. coli. To this end, we determined the maximal copper concentration that E. coli tolerates without any effect on growth to be 1 mM (Fig. S4B), which is much higher than the one found for L. pneumophila (Fig. S4A). Analysis of the lciE-lacZ construct in E. coli resulted in gradual induction of lciE expression as the concentration of copper increased (Fig. 3D). Moreover, this induction was completely dependent on the E. coli CusRS TCS (Fig. 3D). Interestingly, when lciE-lacZ expression was examined in the cusC deletion mutant (CusC together with CusFBA form a copper transporter located in the E. coli envelope [59,61]), higher levels of expression of lciE were observed at all copper concentrations examined (Fig. 3D). This result was obtained probably because E. coli lacking a functional CusCFBA system is exposed to higher concentrations of copper, which led to FIG 2 Two-component systems homologous to lpg0714-lpg0715 are present in many bacteria and are located next to genes encoding metal resistance systems. Schematic representation of genomic regions containing TCSs homologous to lpg0714-lpg0715 found in different bacteria. The homologous response regulators (RRs) are shown in dark blue, the homologous sensor histidine kinases (SHKs) are shown in light blue, the effector lpg0716 is shown in red, and the genes involved in copper resistance, or in resistance to other metals, are shown in gray (most of these genes are not homologous to one another). The position of the conserved regulatory element predicted to be recognized by the lpg0715 RR is shown in purple (Fig. S3). The different designations of the homologous RRs and SHKs are indicated.
The Legionella Copper Island ® higher expression of lciE-lacZ. Comparing this result to the one obtained with the L. pneumophila lciE deletion mutant (compare Fig. 3B and D), which was induced similarly to wild-type L. pneumophila at all copper concentrations, indicates that in L. pneumophila, LciE is not involved in copper transport when the bacteria are grown in media, as expected from an Icm/Dot effector protein that functions during infection of host cells. Furthermore, the higher concentrations of copper tolerated by E. coli led us to examine the copper induction of the L. pneumophila LciRS TCS using E. coli as an in vivo heterologous system. Examination of the lciRS-lciE-lacZ construct in an E. coli cusR deletion mutant indicated that the L. pneumophila LciRS functions in E. coli and can respond to higher concentrations of copper than those tolerated by L. pneumophila (Fig. 3E). Collectively, these results indicate that the CusRS and LciRS TCSs function similarly, and both activate the expression of lciE in response to copper exposure.
LciR recognizes a conserved regulatory element located upstream of lciE. We identified a conserved regulatory element located upstream of lciE and other genes known or expected to be regulated by LciRS homologous TCSs ( Fig. 2 and Fig. S3). This regulatory element constitutes a 7-bp inverted repeat (ATTACAAnnTTGTAAT) as well as two shorter (4 bp each) conserved sequences located between the inverted repeat and the lciE Ϫ10 promoter element ( Fig. 4A and Fig. S3). Examination of the L. pneumophila genomic sequence for the presence of additional such regulatory elements revealed that this site is not present elsewhere in the genome, which is in line with our observation that the LciRS-LciE genomic island was acquired by HGT. To determine the importance of each of the putative lciE regulatory elements for the copper induction by LciR, they were mutagenized and examined for their levels of expression before and after copper induction. Three of these mutated lciE-lacZ fusions completely lost their ability to be induced by copper, and the fourth mutant retained a very limited ability to respond to copper (Fig. 4B).
Since the lciR gene that encodes the RR and the lciE EEG share an intergenic region (Fig. 4A), we examined whether the lciR gene is activated by copper and whether it is an autoregulator. The lciR gene was neither induced by copper nor affected by the lciR deletion mutant (Fig. S6B). These results indicate that the LciRS TCS specifically activates the expression of lciE in response to copper and suggest that the two conserved short regulatory elements located between the inverted repeat and the Ϫ10 promoter element of lciE play a critical role in directing the activation by LciR to lciE and not to lciR.
The L. pneumophila LciR protein directly binds to the regulatory region of lciE. To further support the results presented, the L. pneumophila LciR protein was His tagged, overexpressed, purified, and used for gel mobility shift assays with the lciE regulatory region. The L. pneumophila His 6 -LciR protein was found to bind to the The Legionella Copper Island ® regulatory region of the lciE gene, as evidenced by a shift in the migration of the DNA probe (Fig. 4C). The band shift degree as well as the amount of the shifted probe correlated with the increasing amounts of His 6 -LciR (Fig. 4C). In addition, competition with an unlabeled probe reduced the band shift (Fig. 4C, compare lanes 5 and 6). To further validate the specificity of the binding, we performed additional competition assays with an unlabeled probe containing a mutation in the LciR binding site. When the unlabeled mutated probe was used, a dramatic decrease in competition was observed compared to that of the unlabeled wild-type probe (Fig. 4C, compare lanes  6 and 7).
The mobility shift assay (Fig. 4C), together with the examination of lciE gene expression in the lciR and lciS deletion mutants (Fig. 3) and the analysis of the mutations in the LciR consensus sequence (Fig. 4B), establish LciR as a direct regulator of the lciE EEG in L. pneumophila.
Identification of amino acids required for LciS copper sensing. Even though the E. coli CusRS and the L. pneumophila LciRS TCSs are homologous and both respond to elevated copper concentrations (Fig. 3), the periplasmic sensing domain of CusS and LciS are considerably different in size (150 amino acids in E. coli compared to 28 amino acids in L. pneumophila) and show no sequence homology (Fig. 5A). To identify amino acids required for copper induction in the L. pneumophila LciS periplasmic sensor domain, we aligned the sequences of LciS from the nine Legionella species harboring it. Since the amino acids histidine, cysteine, and methionine were previously shown to be involved in copper binding (62,63), we specifically looked for these residues. Several histidine residues and a single cysteine were found in the short LciS periplasmic domain, together with other conserved amino acids (Fig. 5A). To examine the importance of these conserved amino acids for copper sensing by LciS, seven amino acid residues in the periplasmic domain of LciS (H43, H44, H46, N48, E50, H51, and C60) were changed separately to alanine residues. These mutations were introduced into the lciRS-lciE-lacZ fusion and used to determine the levels of copper induction (Fig. 5B). The most striking result was obtained with the mutation of the cysteine at position 60, which completely abolished the induction by copper. In addition, two of the histidine residues mutated (H44 and H51) significantly reduced induction by copper. We noticed that the number of histidine residues varies between the periplasmic sensor domains of the different Legionella LciS proteins, but at least two histidine residues were present in each LciS periplasmic domain. Thus, we constructed a triple mutation in which the three adjacent histidine residues (H43, H44, and H46) all were changed to alanine residues. This combined mutation resulted in a complete lack of induction by copper (Fig. 5B). To determine the specificity of LciS, we also exposed the bacteria to other similar metals (57,64), and as seen in Fig. 5C, the LciRS TCS was found to be specific to copper and no induction was obtained with any of the other metals examined. Collectively, these results indicate that the Legionella LciS small periplasmic domain contains few histidine residues and a single cysteine residue specifically required for copper sensing.
The LciRS-LciE genomic island is regulated by the Fis repressors. It was previously shown in S. enterica as well as in other bacteria that genomic islands that undergo HGT are silenced by nucleoid-associated proteins (NAPs), such as H-NS and Hha (65)(66)(67). Since the LciRS-LciE island undergoes HGT in the Legionella genus ( Fig. 1 and Fig. S1 and S2), we were interested in examining whether this island is silenced by NAPs. The H-NS NAP is not present in Legionella, but three Fis paralogs (Fis1, Fis2, and Fis3), which are also NAPs, were previously shown to directly repress the expression of EEGs (32). Examination of the lciRS-lciE intergenic region led to the identification of four potential Fis regulatory elements (TG-N 13 -C), two close to or overlapping the Ϫ10 promoter element of lciE (Fig. S7) and two others close to or overlapping the Ϫ10 promoter element of lciR ( Fig. 6A and Fig. S7). To determine whether the Fis repressors are involved in the regulation of the LciRS-LciE island, the expression of the lciE-lacZ fusion was examined in deletion mutants of each of the three fis genes. In the absence of copper, the expression levels of the lciE-lacZ fusion in the three fis deletion mutants were similar to those in the wild-type strain (Fig. 6B). However, in the presence of copper, the expression of the lciE-lacZ fusion was significantly higher in the fis1 and fis3 deletion mutants (Fig. 6B). In addition, when the expression of the lciR-lacZ fusion was examined in the same mutants under the same conditions, there was an increase in the level of expression of the lciR-lacZ fusion in the fis1 deletion mutant that was independent of the presence of copper (Fig. 6C). These results indicate a direct repression of Fis on lciE, on lciR, or on both genes.
Fis1 and Fis3 repress the expression of lciR and affect the copper induction of lciE. To distinguish between the three possibilities described above, we constructed site-directed mutations in the four described putative Fis regulatory elements (Fig. 6A  and Fig. S7). The two mutations constructed in the putative Fis regulatory elements of lciE did not affect its level of expression, with or without copper (Fig. 6D). However, both mutations constructed in the putative Fis regulatory element of lciR (Fig. 6A) showed a relief of repression (Fig. 6E). The mutation in the Fis regulatory element The Legionella Copper Island ® located immediately downstream to the Ϫ10 promoter element of lciR (Fig. 6A) showed a 6-fold relief of repression, and the mutation in the upstream Fis site showed 2-fold relief of repression. A combined mutation in both Fis regulatory elements led to a level of expression similar to that of the mutation in the downstream Fis site (Fig. 6E). It is important to note that the Fis regulatory element located downstream to the lciR Ϫ10 promoter element is conserved in all nine Legionella species harboring the LciRS-LciE genomic island, while the three other putative Fis regulatory elements examined are not conserved (Fig. S7). The effect observed on the level of expression of the lciR-lacZ The lciR and lciE Ϫ10 promoter elements are in dark blue, and the nucleotides representing the LciR consensus are in purple (the inverted-repeat sequence) or light blue (the two sequences located between the inverted repeat and the Ϫ10 promoter of lciE); the inverted repeat is also marked with arrows. The transcription start sites are boldface and underlined. The putative lciR Fis regulatory elements are shaded in yellow, conserved nucleotides of the Fis consensus are marked in red, and the nucleotides mutated are marked by asterisks. (B and C) The expression levels of the lciE-lacZ fusion (B) and the lciR-lacZ fusion (C) were examined in the wild-type strain and in the three fis deletion mutants at the stationary phase. Expression was examined with (gray bars) and without (white bars) 50 M copper. The levels of expression of the lacZ fusions were found to be significantly different (*, P Ͻ 10 Ϫ5 , paired Student's t test) between expression of the wild-type strain and each fis deletion mutant under the same conditions. (D and E) The levels of expression of wild-type lciE-lacZ fusion and the two lciE-lacZ fusions containing mutations (mut-1 and mut-2) in the putative Fis regulatory elements (D) and wild-type lciR-lacZ fusion and the three lciR-lacZ fusions containing mutations (mut-1, mut-2, and mut-1 ϩ 2) in the putative Fis regulatory elements (E) were examined with (gray bars) and without (white bars) 50 M copper. The levels of expression of the lacZ fusions were found to be significantly different (*, P Ͻ 10 Ϫ5 , paired Student's t test) between fusions containing the wild-type regulatory region and the mutated regulatory region under the same copper concentrations.  Fig. 6F, the level of expression of the lciRS-lciE-lacZ fusion containing the mutation in the lciR regulatory element was much higher in the absence of copper (6-fold) as well as in the presence of copper compared to that of the wild-type fusion. Collectively, these results indicate that the repression mediated by Fis on the LciR regulator affects the level of expression of the LciE effector with and without copper, showing that Fis proteins silence this genomic island by repressing the expression of the positive regulator LciR.

DISCUSSION
The L. pneumophila Icm/Dot secretion system translocates into host cells the largest number of effectors known in a single bacterium, and these effectors manipulate numerous host cell processes for the benefit of the bacteria (10)(11)(12)(13)(14). One of the challenges encountered by bacteria using such a multicomponent pathogenesis system is the coordination of the expression of the pathogenesis genes encoding these components to result in a successful infection. Thus far, several regulatory systems that control the expression of EEGs were identified in L. pneumophila (Fig. 7), including a pair of TCSs (PmrAB and CpxRA) that belong to the wHTH family of transcriptional regulators (68). Here, we described a third L. pneumophila TCS (LciRS) that belongs to the wHTH family of RRs, which directly regulates the expression of a single EEG (Fig. 7). In contrast to PmrA and CpxR, LciR is present only in several Legionella species and undergoes HGT as part of a genomic island together with the single EEG it regulates, representing the first case of local regulation of Icm/Dot effectors in Legionella.
One of the most interesting findings regarding the LciRS-LciE genomic island is that The Legionella Copper Island ® it undergoes HGT in the Legionella genus ( Fig. 1; see also Fig. S1 and S2 in the supplemental material). Two aspects regarding this genomic island were left unresolved, namely, (i) the way by which it is transferred between Legionella species and (ii) the way by which it integrates into the bacterial genome. A hint regarding these two issues was obtained when we analyzed the LciRS-LciE genomic island found in two uncharacterized Legionella species (Legionella sp. strain 13.8642 and Legionella sp. strain FW215). In Legionella sp. strain 13.8642, a pseudogene was found to be located next to the LciRS-LciE genomic island (Fig. 1). This pseudogene contains a deletion of a single nucleotide after nucleotide 41, making the protein it used to encode nonfunctional. However, prior to its pseudogenization, this gene encoded a protein with a high degree of homology to a phage integrase. Homologous integrase-encoding genes were found intact in several Legionella species (but not next to the LciRS-LciE genomic island) and in Legionella sp. strain 13.8642 it is completely intact, excluding the single-nucleotide deletion described above. It is possible that this integrase was involved in the integration of the LciRS-LciE genomic island in this Legionella species. Integrase-encoding genes, as well as mutated integrase-encoding genes, were previously shown to be located near genomic islands in other bacteria (69,70). Another known feature of genomic islands is that they sometimes integrate into bacterial genomes next to tRNA genes (50,71). In this case too, in one of the uncharacterized Legionella species, Legionella sp. strain FW215, the LciRS-LciE genomic island was found in proximity to a Lys tRNA gene (Fig. 1). This indicates that tRNA genes are also an entry site for the LciRS-LciE genomic island in Legionella. Although the precise mechanism by which the LciRS-LciE genomic island undergoes HGT in bacteria is not known, the abovementioned findings suggest that it utilizes transfer and integration mechanisms similar to the ones previously described for genomic islands in other bacteria. The LciRS TCS was found to be activated in the presence of copper (Fig. 3), and homologous TCSs present in other bacteria are also activated by copper and are usually located next to genes involved in metal resistance (mainly copper) (59). Comparison of the Legionella LciRS TCS to the homolog TCSs present in other bacteria resulted in the identification of a major difference: the Legionella SHK LciS was found to contain a small (28 amino acids long) (Fig. 5A) periplasmic sensor domain, which is completely nonhomologous to the periplasmic sensing domains found in the homologous SHKs present in other bacteria, including the ones that sense copper. This finding is intriguing, since SHKs were previously shown to change their sensing domain by recombination or mutations and, in this way, change the signal they respond to and deviate from other SHKs (72). However, in the case of LciS, the sensing domain was completely changed but the signal recognized by the SHK remained the same. One can speculate on the evolutionary driving forces that can lead a sensing domain to be replaced without changing the signal it responds to. For example, an advantage can be achieved if the new sensing domain alters the sensitivity or specificity to the signal. However, examination of these two aspects, by comparing the E. coli CusS and L. pneumophila LciS SHKs, did not result in differences in the sensitivity to copper (compare Fig. 3B and D) or in the specificity to copper (Fig. 5C and data not shown). Even though the LciS periplasmic sensor domain is not homologous to other sensing domains that sense copper, it is important to mention that we did recognize specific amino acids critical for the sensing of copper by the L. pneumophila LciS, and the same amino acids were previously shown to be involved in copper binding in different proteins (62,63).
Genes regulated by regulatory systems that sense a specific signal were shown to encode proteins with functions directly related to the signal sensed by their regulators. This phenomenon was demonstrated in many systems, including regulators of amino acids biosynthetic pathways and regulators of metal resistance systems (59,73). In addition, the L. pneumophila mavN EEG, which encodes an iron transporter localized to the LCV, is regulated by the iron-specific repressor Fur (33,34). However, in many cases the signal sensed by a regulator is not directly related to the function mediated by the genes it regulates. In these cases, the signal sensed indicates a change in the bacterial environment. Such cases were described in many pathogenesis systems, such as the S. enterica PhoPQ TCS, which senses Mg 2ϩ and cationic antimicrobial peptides (74,75), and the S. enterica PmrAB TCS, which senses acidic pH and high Fe 3ϩ concentrations (76,77). Both of these TCSs regulate the expression of numerous genes encoding diverse functions (78,79). The function of LciE, which is activated by the LciRS TCS in response to copper, is currently unknown, and it contains no known protein domains. However, we identified two other L. pneumophila effectors with unknown functions that show a significant degree of homology to LciE (Fig. S8). The core effector CetLp1 (lpg0140), and another effector (lpg2888) that is found in most of the Legionella species examined, harbor a protein domain homologous to LciE (Fig. S8). In addition, these three effectors contain four predicted transmembrane domains located at the C-terminal half of the protein (Fig. S8). These similarities among the three effectors suggest that they perform a related function. Since both copper and zinc are used by eukaryotic cells to kill invading bacteria (80,81), we examined the copper-or zincdependent induction of CetLp1 and lpg2888, or an effect of LciR on their levels of expression, but their expression was unaffected ( Fig. S9A and B). Moreover, we generated a triple deletion mutant of cetLp1, lpg288, and lciE and examined this mutant using a competition assay in amoeba with and without the addition of copper, and no intracellular growth phenotype was observed ( Fig. S9C to F).
Unlike all the regulatory systems described thus far for L. pneumophila effectors, the LciRS-LciE genomic island represents a new type of effector regulation, unprecedented in the Legionella genus. In this system, a single effector is locally regulated by a dedicated regulatory system in response to a specific signal. Moreover, both the regulatory system and the effector form a unit that undergoes HGT in the Legionella genus. Uncovering this novel type of regulation sheds new light on the ways the effector repertoire of Legionella evolves and the activating signals of effectors expand.

MATERIALS AND METHODS
Bacteria strains and media. The L. pneumophila wild-type strain used in this work was JR32, a streptomycin-resistant, restriction-negative mutant of L. pneumophila Philadelphia-1, which is a wild-type strain in terms of intracellular growth (82). In addition, mutant strains derived from JR32 that were used in this study are listed in Data Set S1 in the supplemental material. The E. coli strains used in this work are also listed in Data Set S1. Bacterial media, plates, and antibiotics were as previously described (83).
Plasmid construction. To construct lacZ translational fusions (Data Set S1), the 300-bp regulatory regions of the lciE and lciR genes were amplified by PCR using the primers listed in Data Set S1. The PCR products were then digested with BamHI and EcoRI, cloned into pGS-lac-02, and sequenced. The lciE lacZ fusion, which does not contain the lciRS genes, was designated lciE-lacZ. In addition, a second lciE lacZ fusion was constructed containing the lciRS genes in an organization similar to that in the genome. To construct this fusion, an internal EcoRI site present in the lciR gene was mutagenized in a way that does not change the LciR amino acid sequence. A 1,950-bp region was amplified by PCR using the four primers listed in Data Set S1, digested with BamHI and EcoRI cloned into pGS-lac-02, and sequenced. This lciE lacZ fusion, which contains the lciRS genes, was designated lciRS-lciE-lacZ.
To construct a substitution mutation in the putative LciR binding site in the regulatory region of the lciE gene, a substitution mutation in the putative Fis binding site in the regulatory region of the lciE and lciR genes, and substitution mutations in the lciR and lciS coding sequence, site-directed mutagenesis was performed by regular PCR or the PCR overlap extension approach (84), as previously described (19). The primers used for the mutagenesis are listed in Data Set S1, and the plasmids resulting from the site-directed mutagenesis are listed in Data Set S1.
To construct deletion substitution mutants in the L. pneumophila lciE and lciS genes, a 1-kb DNA fragment located on each side of the planned deletion was amplified by PCR using the primers listed in Data Set S1. The resulting plasmids were digested with the suitable enzymes, and the inserts were used for a four-way ligation containing the Km resistance cassette (Pharmacia). The plasmids generated, pAA-lpg0714-Km and pMLpUC18ϩ0716Up-Km-Dw (Data Set S1), were digested with PvuII, and the resulting fragment was cloned into the pLAW344 allelic exchange vector digested with EcoRV to generate the plasmids pAA-lpg0714-pLAW and pMLpLAW344-0716-Up-Km-Dw (Data Set S1). In addition, the insert of pMLpUC18ϩ0716Up-Km-Dw was also cloned into the pGY100 allelic exchange vector, digested with XmnI, to generate the plasmid pMLpGY100ϩlpg0716-Up-Km-Dw, which was later digested with SalI to take out the Km resistance cassette and generate the plasmid pMLpGY100ϩlpg0176-UPϩDw (Data Set S1). The latter plasmid was used to generate a clean lciE deletion.
To generate the triple lciRS-lciE deletion mutant, the insert containing the 1-kb upstream region of lciS and the insert containing the 1-kb downstream region of lciE were used for a four-way ligation, as described above, to generated plasmid pMLpUC18ϩ0714Dw-Km-0716Dw (Data Set S1). This plasmid was digested with PvuII, and the resulting fragment was cloned into the pLAW344 allelic exchange vector digested with EcoRV to generate the plasmid pMLpLAW344-0714Dw-Km-0716Dw (Data Set S1). To generate a clean deletion mutant in lpg2888, a 1-kb DNA fragment located on each side of the planned deletion was amplified by PCR using the primers listed in Data Set S1. The resulting plasmids were digested with the suitable enzymes, and the inserts were used for a three-way ligation into pUC-18 to generate pMW-18-Δlpg2888-3W (Data Set S1). This plasmid was digested with PvuII, and the resulting fragment was cloned into the pGY100 allelic exchange vector digested with XmnI to generate the plasmid pMW-100-Δlpg2888 (Data Set S1). The clean and marked allelic exchange deletion mutants were constructed as previously described (83,85).
For the construction of the plasmid expressing the His-tagged LciR, the lciR gene was amplified by PCR using the primers listed in Data Set S1, cloned into pET-15b, and sequenced to generate the plasmid pML-pET15bϩlpg0715 (Data Set S1).
Bacterial growth in the presence of copper. To determine the copper concentrations to be used in the ␤-galactosidase assays with both L. pneumophila and E. coli, the bacteria were grown in fresh AYE lacking Fe(NO 3 ) 3 or LB, respectively, with a wide range of copper concentrations, and the optical density at 600 nm (OD 600 ) was determined in intervals of 1 h until reaching stationary phase. The same analysis was also performed with the other metals examined.
␤-Galactosidase assay. ␤-Galactosidase assays were performed as previously described (19). L. pneumophila strains were grown for 48 h on charcoal-yeast extract (CYE) plates containing chloramphenicol (Cm). The bacteria were scraped off the plate and suspended in ACES-yeast extract (AYE) broth, and the bacterial OD 600 was calibrated to 0.1 in fresh AYE lacking Fe(NO 3 ) 3 , containing different concentrations of copper (or other metals, when indicated) and Cm. When other metals were used, 2 M bathocuproine sulfonate (BCS) was added to the medium to adsorb any copper traces present in the stocks of the other metals. This concentration of BCS was also included when copper was used, and it did not affect the induction by copper. The resulting cultures were grown on a roller drum for about 18 h, until reaching an OD 600 of about 3.2 (early stationary phase), and used for ␤-galactosidase assay.
␤-Galactosidase assays in E. coli were performed similarly, but the E. coli strains were grown for about 6 h in LB containing different concentrations of copper, until reaching an OD 600 of about 2.5 (early stationary phase), and used for ␤-galactosidase assay. The assays were done for 20, 50, or 100 l of culture, and the substrate for ␤-galactosidase hydrolysis was o-nitrophenyl-␤-D-galactopyranoside.
Protein purification and gel mobility shift assay. His 6 -LciR was purified from E. coli BL21(DE3) using nickel bead columns (Qiagen) according to the manufacturer's instructions. After purification, the fractions containing the protein were dialyzed overnight against a buffer containing 10 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 50 mM KCl, 0.1 mM EDTA, and 0.1 mM dithiothreitol. Glycerol was added to a concentration of 50%, and the purified protein was then stored at -20°C. A gel mobility shift assay was performed as previously described (32), with a few modifications. The regulatory region lciE (176 bp) was amplified by PCR using the primers listed in Data Set S1 and 3= end labeled with digoxigenin (DIG) by using DIG-11-ddUTP (Roche). Increasing amounts of the purified His 6 -LciR protein (between 35 and 280 nM) were mixed with 0.75 nM the DIG-labeled probe in buffer containing 10 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 50 mM KCl, 0.1 mM EDTA, 0.1 mM dithiothreitol, 250 g/ml bovine serum albumin, and 50 g/ml herring sperm DNA. For the competition experiments, a 100-fold excess of the unlabeled probe or mutated unlabeled probe was allowed to bind the His 6 -LciR protein for 30 min before addition of the DIG-labeled probe. The binding reaction was carried out for 30 min at room temperature, and samples then were loaded onto a 5% polyacrylamide-0.25ϫ Tris-acetate-EDTA gel in 0.5ϫ Tris-acetate-EDTA running buffer. Following electrophoresis, the gel was transferred to a nylon membrane and fixed by UV cross-linking. The DIG-labeled DNA fragments were detected by following the manufacturer's instructions (Roche). Intracellular growth assays. Intracellular growth assays of L. pneumophila strains in A. castellanii and HL-60-derived human macrophages were performed as previously described (86). Intracellular competition assays of L. pneumophila strains in A. castellanii also were performed as previously described (22).
Reconstruction of phylogenetic trees. Trees were reconstructed on the basis of concatenated alignments of the three proteins indicated for each tree. The trees were reconstructed using RAxML (87) under the LG ϩ GAMMA evolutionary model with 100 bootstrap resampling.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.