Analysis of DNA Regulatory Elements Required for … of DNA Regulatory Elements Required for...

JOURNAL OF BACTERIOLOGY, July 2002, p. 3823–3833 Vol. 184, No. 140021-9193/02/$04.00�0 DOI: 10.1128/JB.184.14.3823–3833.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Analysis of DNA Regulatory Elements Required for Expression ofthe Legionella pneumophila icm and dot Virulence Genes

Ohad Gal-Mor, Tal Zusman, and Gil Segal*Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences,

Tel Aviv University, Ramat-Aviv, Tel Aviv 69978, Israel

Received 7 March 2002/Accepted 22 April 2002

To investigate the regulation of the Legionella pneumophila icm and dot genes required for intracellulargrowth, a series of nine icm::lacZ fusions were constructed. These icm::lacZ fusions were found to have differentlevels of expression in L. pneumophila, and five of them were more highly expressed at stationary phase thanat exponential phase. When the expression of these fusions in Escherichia coli was tested, all of them were foundto be expressed but three of them had dramatic changes in their levels of expression in comparison to thosein L. pneumophila. Site-directed and PCR random mutagenesis with these icm::lacZ fusions was used to identifyDNA regulatory elements of icm genes. Four icm genes (icmT, icmP, icmQ, and icmM) that had low levels ofexpression in L. pneumophila were found to contain a 6-bp sequence (TATACT) essential for their expression.This sequence was shown by primer extension to serve as their �10 promoter elements. A similar sequence,which constitutes the �10 promoter elements of the icmV, icmW, and icmR genes which had high levels ofexpression in L. pneumophila, was also identified. In addition, regulatory elements that probably serve asbinding sites for transcription regulators were found in these genes. Altogether, 12 regulatory elements, 7 ofwhich constitute the �10 promoter elements of the icm genes, were found. Even though all the icm and dot genesare part of one system required for L. pneumophila intracellular growth and even though their promoters areprobably recognized by the vegetative sigma factor, it seems that they are subjected to different regulationmediated by several regulatory factors.

Legionella pneumophila, the causative agent of Legionnaires’disease, is a facultative intracellular pathogen. L. pneumophilais able to infect, multiply within, and kill human macrophages,as well as free-living amoebae (28, 38). The bacteria are takenup by regular phagocytosis or by a special mechanism termed“coiling” phagocytosis (9, 26). They are then found within aspecialized phagosome that does not fuse with lysosomes oracidify (9, 24, 27). The specialized phagosome undergoes sev-eral recruitment events, which include association with smoothvesicles, mitochondria, and the rough endoplasmic reticulum(1, 25, 49); the bacteria multiply within the specialized phago-some until the cell eventually lyses, releasing bacteria that canstart new rounds of infection (28, 38).

Two regions of genes required for human macrophage kill-ing and intracellular multiplication have been discovered in L.pneumophila (reviewed in references 44 and 51). Region Icontains 7 genes (icmV, -W, and -X and dotA, -B, -C, and -D)(8, 10, 32, 50), and region II contains 17 genes (icmT, -S , -R,-Q, -P, -O, -N, -M, -L, -K, -E, -G, -C, -D, -J, -B, and -F) (3, 35,41, 43, 50). Complementation analysis of these genes indicatedthat they are probably organized as nine transcriptional units(icmTS, icmR, icmQ, icmPO, icmMLKEGCD, icmJB, icmF-tphA, icmWX, and icmV-dotA) (8, 10, 35, 41, 43, 50). Most ofthese genes were also shown to be required for intracellulargrowth in protozoan host Acanthamoeba castellanii (45). Four-teen of the Icm and Dot proteins (IcmT, -P, -O, -L, -K, -G, -C,-D, -J, and -B and DotA, -B, -C, and -D) were found to have

significant sequence similarity to Tra and Trb proteins fromIncI plasmids colIb-P9 and R64 (29, 46). The icm/dot system isbelieved to serve as a translocation system that delivers effectorproteins into host cells (34, 44).

At the present time there is very little information about theregulation of L. pneumophila virulence, as well as about regu-latory factors that control the expression of the 24 icm and dotgenes. So far, stationary-phase sigma factor rpoS has beenshown to be involved in L. pneumophila virulence: a strain witha knockout in this gene lost its ability to grow in protozoan hostA. castellanii (20) and was attenuated for intracellular growthin murine bone marrow-derived macrophages (4). However,this gene was found to be dispensable for growth in HL-60-derived human macrophages and in THP-1 cells (20). Anotherstationary-phase-related factor whose involvement in the reg-ulation of L. pneumophila virulence was suggested was the relAgene product (21), but this gene was found to be dispensablefor intracellular growth (55). In addition, the expression ofonly one out of nine icm::lacZ fusions was reduced in strainscontaining an insertion in the rpoS or the relA gene (55),indicating that other factors control the expression of the icmand dot genes.

We were interested in identifying DNA regulatory elementsthat control icm and dot gene expression. To address this goal,we used random and site-directed mutagenesis on the regula-tory regions of nine icm and dot genes that were fused to thelacZ reporter. We identified 12 regulatory elements in theupstream region of eight icm and dot genes. Primer extensionanalysis indicated that seven of these sites constitute the �10promoter elements of the icm genes; the other sites are ex-pected to serve as binding sites for transcription regulators.

* Corresponding author. Mailing address: Department of MolecularMicrobiology and Biotechnology, George S. Wise Faculty of Life Sci-ences, Tel Aviv University, Ramat-Aviv, Tel Aviv 69978, Israel. Phone:972-3-6405287. Fax: 972-3-6409407. E-mail: [email protected].

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MATERIALS AND METHODS

Bacterial strains, plasmids, primers, and media. The L. pneumophila strainused in this work was JR32, a streptomycin-resistant, restriction-negative mutantversion of L. pneumophila Philadelphia-1, which is a wild-type strain in terms ofintracellular growth (39). The Escherichia coli strain used was MC1061 with adeletion of the lacZ gene (13). Plasmids and primers used in this work aredescribed in Tables 1 and 2, respectively. Bacterial media, plates, and antibioticconcentrations used were as described before (43).

Plasmid construction. Promoterless lacZ vector pGS-lac-01 (55) was used toconstruct promoterless lacZ vector pGS-lac-02. Vector pGS-lac-01 contains twoBamHI sites, one in the region immediately upstream from the promoterlesslacZ gene and another downstream from the lacZ gene. To use the BamHI sitefor cloning, pGS-lac-01 was partially digested with BamHI and self ligated afterfill-in. One of the plasmids generated in which the BamHI site located upstreamfrom the lacZ gene was left unchanged and the second site was diminished wasnamed pGS-lac-02.

The plasmids that were used as templates for the sequencing reactions ana-lyzed together with the primer extension reactions are listed in Table 1. PlasmidpGS-Lp-82 was constructed by cloning a HindIII-EcoRV fragment containingthe icmW gene, part of the icmV and icmX genes, and the regulatory regionbetween icmV and icmW into the pUC-18 vector.

Site-directed mutagenesis. Site-directed mutagenesis was performed by theoverlap extension PCR method (23). For each mutation, two primers that con-tain the mutation and that overlap one another by 20 bp were designed. The PCRtemplate for all the mutations was the regulatory region of the gene of interestcloned in the pMC1403 vector (Table 1). PCR mutagenesis includes two steps. Inthe first step two PCR fragments were generated with the following pairs ofprimers: (i) a primer located on the vector, upstream from the regulatory region(pMC-amp), and one of the primers that contain the mutation and (ii) a primerlocated in the lacZ gene on the vector (pMC-lac) and the other primer thatcontains the same mutation on the complementary strand. The resulting twofragments were gel purified and used as templates in the second step, whichincludes a third PCR using the two primers located on the vector. The resultingPCR product was digested with BamHI and cloned into the pGS-lac-02 vector.All the mutations were confirmed by sequencing the whole regulatory region.The changes made were always A to C, T to G, and C to A.

PCR random mutagenesis. Random PCR mutagenesis was performed essen-tially as described before (18). It was performed on the icmF regulatory regionby using the icmF reverse primer containing a BamHI site (icmF-Bam) and theicmF forward primer containing an EcoRI site (icmF-EI) (Table 2). The PCRmixture included either 50 mM dATP, 0.5 mM MnCl2, and 9.5 mM MgCl2 or 50mM dCTP, 0.5 mM MnCl2, and 5.5 mM MgCl2, along with other deoxynucleo-

side triphosphates at 0.2 mM, 100 ng of template (pGS-reg-F2), 25 pmol of eachprimer, and 1 U of Super-Therm DNA polymerase. PCR under these conditionsresults in 1 to 3 bp changes per 100 bp of amplicon. The PCR product wasdigested with EcoRI and BamHI and cloned into the pGS-lac-02 vector digestedwith the same enzymes. The resulting colonies were screened on MacConkeyplates, suspected colonies were isolated, and the mutated regulatory regionsfrom some of them were sequenced.

�-Galactosidase assays. �-Galactosidase assays were performed as describedelsewhere (33). L. pneumophila strains were grown on ABCYE (ACES bufferedcharcoal yeast extract) plates containing chloramphenicol for 48 h. The bacteriawere scraped off the plate and suspended in AYE (ACES yeast extract) broth,and bacterial optical density at 600 nm (OD600) was calibrated to 0.1 in AYE.The resulting cultures were grown on a roller drum for about 18 h until reachingan OD600 of about 3.8 (stationary phase). To test the levels of expression atexponential phase, the cultures were diluted to an OD600 of 0.1 and grown for anadditional 6 to 7 h until reaching an OD600 of about 0.7 (exponential phase). Theassays were done with 50 or 100 �l of culture. The substrate for lacZ hydrolysiswas o-nitrophenyl-�-D-galactopyranoside.

Preparation of RNA. RNA preparation was performed as described elsewhere(42). The L. pneumophila JR32 strain was grown on ABCYE plates for 48 h. Thebacteria were scraped off the plate and suspended in AYE broth, and thebacterial OD600 was calibrated to 0.1 in AYE. The resulting cultures were grownon a roller drum for about 18 h until reaching an OD600 of about 3.8. Then thecultures were diluted to OD600 of 0.1 in AYE and grown for additional 6 to 7 huntil reaching an OD600 of about 0.7. These bacteria were used for RNA prep-aration. The cell pellets derived from 30 ml of bacteria were suspended in 3 mlof STE buffer (10 mM Tris [pH 7], 100 mM NaCl, 1 mM EDTA), and an equalvolume of aqueous 95% hot phenol (65°C) was added. This mixture was shakenvigorously at 65°C for 10 min and centrifuged for 10 min at 11,000 � g. Theaqueous phase was removed, and the phenol phase was reextracted with 3 ml ofSTE buffer. After centrifugation, the aqueous phase was removed and combinedwith that already collected, and the solution was extracted twice with hot phenol,once with phenol-chloroform-isoamyl alcohol (25:24:1), and twice with STE-saturated ether. The RNA was precipitated with 2 volumes of ethanol. Afterapproximately 12 h of incubation at �20°C, the precipitate was recovered bycentrifugation at 11,000 � g for 10 min. The RNA concentration was determinedby measuring OD260.

Primer extension analysis. The primers used for the analysis are listed in Table2. A reverse transcriptase (RT) reaction was performed in RT buffer (50 mMTris-Cl [pH 8.3], 8 mM MgCl2, 40 mM KCl, 1 mM dithiothreitol) containingdeoxynucleoside triphosphates (1 mM concentration [each] of dATP, dCTP,dGTP, and dTTP), RNasin (10 U), 5 pmol of labeled ([�-32P]ATP) primer, and5 U of avian myeloblastosis virus RT (Promega, Madison, Wis.). A total of 10 �gof the RNA preparation was used in a total reaction mixture volume of 6 �l. Thereaction mixture was incubated for 5 min at 45°C and then incubated at 37°C for30 min. After the addition of 4 �l of sequencing stop buffer, the samples wereheated for 5 min at 90°C before being loaded on a sequencing gel.

DNA sequencing. The sequence was determined by the dideoxy terminationmethod (40) using the SequiTherm cycle sequencing kit (Epicentre, Madison,Wis.). The templates for the sequencing reactions were plasmids containing theregulatory regions of the genes tested (Table 1). Sequencing was determinedwith the same primers used for the primer extension analysis.

RESULTS

We were interested in identifying DNA regulatory elementsthat control the expression of the L. pneumophila icm and dot

TABLE 2. Primers used in this study

Primer Sequence (5�–3�)

PE-icmP ..............GACGATCCATACTGGCGCCATGPE-icmQ..............TGGACCTTTTTCAATAGCATCGPE-icmR..............AAAAACCAAATGGATTTCGTGCACTGPE-icmV..............TTCATTCTGTCCCAATCGAACCPE-icmW.............CCAGTACTTTGCGGAGGCTTCATGGCIcmF-EI...............GCCGGAATTCGAACAAGGAGCAAGTATTTCIcmF-Bam ...........CGGGGGATCCCCGTATTGCTCAGTTGTCATTATATpMC-lac...............TAAGTTGGGTAACGCCAGGGpMC-amp............AAGGGAATAAGGGCGACACG

TABLE 1. Plasmids used in this study

Plasmid Features Referenceor source

pGS-lac-01 pAB-1 with a promoterless lacZ gene 55pGS-lac-02 pAB-1 with a promoterless lacZ gene This studypGS-Lp-34 icmPO and their regulatory region in pUC-18 43pGS-Lp-35 icmQ and its regulatory region in pUC-18 43pGS-Lp-36 icmR and its regulatory region in pUC-18 43pGS-Lp-82 icmV and icmW regulatory region in pUC-18 This studypGS-reg-F2 Regulatory region of icmF in pMC1403 55pGS-reg-F3 Regulatory region of icmF in pGS-lac-01 55pGS-reg-M2 Regulatory region of icmM in pMC1403 55pGS-reg-M3 Regulatory region of icmM in pGS-lac-01 55pKP-Q-21 Regulatory region of icmQ in pMC1403 55pOG-J-109 Regulatory region of icmJ in pMC1403 55pOG-J-122 Regulatory region of icmJ in pGS-lac-01 55pOG-P-108 Regulatory region of icmP in pMC1403 55pOG-P-121 Regulatory region of icmP in pGS-lac-01 55pOG-Q-126 Regulatory region of icmQ in pGS-lac-01 55pOG-R-125 Regulatory region of icmR in pGS-lac-01 55pOG-T-107 Regulatory region of icmT in pMC1403 55pOG-T-120 Regulatory region of icmT in pGS-lac-01 55pOG-V-110 Regulatory region of icmV in pMC1403 55pOG-V-123 Regulatory region of icmV in pGS-lac-01 55pOG-W-111 Regulatory region of icmW in pMC1403 55pOG-W-124 Regulatory region of icmW in pGS-lac-01 55pSS-R-27 Regulatory region of icmR in pMC1403 55pUC18 oriR(ColE1) MCSa Apr 54

a MCS, multiple cloning sites.

3824 GAL-MOR ET AL. J. BACTERIOL.

genes required for intracellular growth. According to comple-mentation analysis that was performed previously (10, 35, 41,43), the icm and dot genes are organized on nine transcrip-tional units (icmTS, icmR, icmQ, icmPO, icmMLKEGCD,icmJB, icmF-tphA, icmWX, and icmV-dotA). The results ob-tained by the complementation analysis are in agreement withthe organization of the genes in the two icm/dot regions. Thereare between 91 and 400 bp of a noncoding region upstream ofgenes expected to be first in a transcriptional unit and between0 and 35 bp of a noncoding region upstream of genes that arenot expected to be first in a transcriptional unit. On the basisof these data, nine icm::lacZ fusions (icmT::lacZ, icmR::lacZ,icmQ::lacZ, icmP::lacZ, icmM::lacZ, icmJ::lacZ, icmF::lacZ,icmV::lacZ, and icmW::lacZ) that contain the whole regionbetween the gene of interest and the gene located upstream ofit, with a minimum of 150 bp, were constructed.

Analysis of the expression levels of icm::lacZ fusions in L.pneumophila. It has been suggested that L. pneumophila patho-genesis is coordinated with entry into stationary phase (4, 11,21). To test whether icm and dot genes that were shown to berequired for intracellular growth are regulated in correlationwith growth phase, we compared the expression levels of thenine icm::lacZ fusions at exponential and stationary phases.

Even though all the icm and dot genes were shown to berequired for intracellular growth and host cell killing, theirexpression levels were found to be different from one another.As can be seen in Fig. 1, the icm::lacZ fusions can be dividedinto two groups according to their expression levels in thewild-type L. pneumophila JR32 strain. Four icm::lacZ fusions(icmR::lacZ, icmF::lacZ, icmV::lacZ, and icmW::lacZ) werefound to have high levels of �-galactosidase expression (above1,000 Miller units [MU]), and two of them had higher levels ofexpression at stationary phase than at exponential phase (Fig.1A). In contrast, five icm::lacZ fusions (icmT::lacZ, icmP::lacZ,icmQ::lacZ, icmM::lacZ, and icmJ::lacZ) were found to havelow levels of �-galactosidase expression (below 1,000 MU),and three of these fusions had higher levels of expression atstationary phase than at exponential phase (Fig. 1B). Only oneof these fusions (icmP::lacZ) was shown to have lower levels ofexpression in strains containing an insertion in the genes cod-ing for RpoS or RelA than in wild-type strains (55); the otherfusions (icmT::lacZ, icmM::lacZ, icmR::lacZ, and icmF::lacZ),which had higher levels of expression at stationary phase thanat exponential phase, are probably regulated by other factors.

Analysis of the expression levels of icm::lacZ fusions in E.coli. One way to get an indication of whether unique L. pneu-mophila regulatory factors participate in icm gene expression isto compare their expression in L. pneumophila with that in E.coli. When we compared the levels of expression of theicm::lacZ fusions described above in E. coli and L. pneumo-phila, several interesting differences were observed (compareFig. 1 and 2). Three icm::lacZ fusions (icmF::lacZ, icmV::lacZ,and icmW::lacZ) that had high levels of expression in L. pneu-mophila were also found to have high levels of expression in E.coli (compare Fig. 1A and 2A). In contrast, the icmR::lacZfusion, which was highly expressed in L. pneumophila, had avery low level of expression in E. coli (fourfold reduction atexponential phase and sevenfold reduction at stationary phase;compare Fig. 1A and 2B). Two other fusions (icmQ::lacZ andicmM::lacZ) had the opposite result: their levels of expression

were higher in E. coli than in L. pneumophila, showing anincrease of at least sevenfold both at exponential and station-ary phases (compare Fig. 1 and 2). Four out of the fiveicm::lacZ fusions that had higher levels of expression at sta-tionary phase than at exponential phase in L. pneumophila(icmT::lacZ, icmP::lacZ, icmM::lacZ, and icmR::lacZ) showedincreased levels of �-galactosidase at stationary phase in E. colias well, but the increase was less pronounced.

We think that these results clearly indicate that the promot-ers of the L. pneumophila icm and dot virulence genes arerecognized in E. coli and are probably transcribed by a sigmafactor present in both bacteria. However, it is most likely thatthere are other regulatory factors (a repressor for icmQ andicmM and an activator for icmR) present in L. pneumophilathat are absent in E. coli.

Identification of �10 promoter elements of icm genes thathave low expression levels. Careful analysis of the upstreamregulatory regions of four icm genes (icmT, icmP, icmQ, andicmM) that were found to have low levels of expression in L.pneumophila, revealed a 6-bp (TATACT) putative consensussequence (Fig. 3A). The distances of this sequence from the

FIG. 1. Expression of icm::lacZ fusions in L. pneumophila at expo-nential and stationary phases. The expression of the nine icm::lacZfusions (for the genes icmT, icmR, icmQ, icmP, icmM, icmJ, icmF,icmW, and icmV) in wild-type strain JR32 at exponential (gray) andstationary phases (white) was examined. �-Galactosidase activity wasmeasured as described in Materials and Methods. Four of theicm::lacZ fusions were found to have high �-galactosidase activities(A), and five were found to have low activities (B). The results (Millerunits [M.U.]) are the averages � standard deviations (error bars) of atleast three different experiments.

VOL. 184, 2002 REGULATORY ELEMENTS OF THE L. PNEUMOPHILA icm GENES 3825

first ATG codons of the corresponding genes (32 to 74 bp), aswell as the sequence itself, suggest that it might serve as the�10 promoter element for these genes. This sequence differsby only 1 nucleotide from the �10 promoter element recog-nized by E. coli vegetative sigma factor RpoD (TATAAT) (53).To examine the importance of this sequence in the expressionof these icm::lacZ fusions, 2 nucleotides in each of these se-quences were mutated and the effect of the change was exam-ined. As can be seen in Fig. 3B, the change in either of thesetwo positions (the second or third nucleotide of the putativeconsensus sequence) caused a dramatic reduction in the levelsof expression of these icm::lacZ fusions. The mutation in thesecond position of the putative consensus (mutation 1 in Fig. 3)almost eliminated the expression of the lacZ reporter, and thelevels of �-galactosidase observed were close to the expressionlevel of the promoterless lacZ vector, the negative control(pGS-lac-02) (data not shown).

As indicated in the two previous sections, icmT and icmPhave similar expression levels and expression profiles in L.pneumophila (higher expression at stationary phase than atexponential phase). In addition, they contain the same 4 nu-cleotides upstream from their TATACT consensus sequences;these 4 nucleotides might form an inverted repeat with the

consensus found (Fig. 3A). On the other hand, icmM and icmQalso have similar levels of expression in L. pneumophila andthey were both found to have higher levels of expression in E.coli than in L. pneumophila (sevenfold increase at both station-ary and exponential phases). Due to these results, the tran-scription start sites for one of the genes in each pair (icmP andicmQ) were determined (Fig. 3A), and the primer extensionresult for the icmP gene is presented in Fig. 3C. As can be seenclearly from the primer extension analysis, the TATACT siteconstitutes the �10 promoter element of these genes, as wasexpected from the distance of the consensus from the firstATG of the genes, the sequence itself, and the mutagenesisresults.

All the positions in the TATACT consensus are required formaximal expression. To determine if all the positions in theTATACT consensus are required for maximal expression, weperformed a careful analysis of the icmQ consensus sequence(Fig. 4). Eight mutations in the icmQ consensus sequence wereconstructed (Fig. 4A): the 6 nucleotides of the TATACT con-sensus as well as 1 nucleotide on both sides of it were mutated.As can be seen very clearly in Fig. 4B, all six positions that areexpected to be part of the consensus sequence were found tobe required for maximal expression of the icmQ::lacZ fusion.The mutations upstream and downstream from the consensussequence did not reduce the expression of the icmQ::lacZfusion, and they determine the boundaries of the consensus.

A primer extension analysis of this region determined thetranscription start site of the icmQ transcript (Fig. 4C). Thetranscription start site was located 6 bp downstream from thelast nucleotide of the consensus, indicating that the TATACTconsensus constitutes the �10 promoter element of this gene.It is important to note that the changes at positions 1, 2, and 6caused the strongest reduction in the expression level of theicmQ::lacZ fusion whereas the changes in the three other po-sitions of the consensus (positions 3, 4, and 5) had a clear butless dramatic effect on the expression of this fusion. Theseresults are in agreement with the known data about E. colirpoD promoters showing that the first, second, and sixth nu-cleotide of its �10 promoter element are the most conserved(31).

icmR contains at least three regulatory elements. Analysis ofthe icmR regulatory region identified two identical sequences(AAGATATATT) located next to each other. Because the 6bp (TATATT) located at the 5� end of this sequence differ byonly 1 nucleotide from the �10 promoter element describedabove (TATACT), we thought that these two sites might serveas regulatory elements of the icmR gene. To test this hypoth-esis, single as well as double mutations in the icmR regulatoryregion were constructed (Fig. 5A). As can be seen in Fig. 5B,the effect of the mutations in the icmR regulatory region wasless pronounced than the effect of mutations in icmT, icmP,icmQ, and icmM. However, the combination of the two muta-tions at the third position (mutation 6 in Fig. 5B) caused areduction of about 50% in the expression of the icmR::lacZfusion, which was a more severe reduction than that due toeach individual mutation at this position (mutations 2 and 4 inFig. 5B).

To determine if either of these two sites serves as a �10promoter element of the icmR gene, the start site of its tran-script was determined (Fig. 5C). The transcription start site

FIG. 2. Expression of icm::lacZ fusions in E. coli at exponential andstationary phases. The expression of the nine icm::lacZ fusions (for thegenes icmT, icmR, icmQ, icmP, icmM, icmJ, icmF, icmW, and icmV) inE. coli strain MC1061 at exponential (gray) and stationary phases(white) was examined. �-Galactosidase activity was measured as de-scribed in Materials and Methods. Five of the icm::lacZ fusions werefound to have high �-galactosidase activities (A), and four were foundto have low activities (B). The results (Miller units [M.U.]) are theaverages � standard deviations (error bars) of at least three differentexperiments.


clearly indicates that the two sites mutated are not the �10promoter element of the icmR gene. The sequence of its �10promoter element was found to be TATATG, which is only 2nucleotides different from the �10 promoter elements identi-fied for icmT, icmP, icmQ, and icmM. We concluded that theAAGATATATT consensus plays a significant regulatory rolein the expression of the icmR gene, probably as a binding sitefor a regulatory protein, and that the TATATG site constitutesthe �10 promoter element of this gene.

Identification of a regulatory elements of icm genes thathave high expression levels. icmW, icmV, and icmF are the onlythree genes that were found to have high levels of expressionin both L. pneumophila and E. coli (Fig. 1A and 2A). To findregulatory elements that are involved in the expression of thesegenes, the regulatory region of the icmF transcriptional unitwas randomly mutated by PCR. Four clones that each containa single mutation were identified, and all mutations mappedclose to one another. Three mutations (from three indepen-dent PCRs) were found at the same position, and a fourthmutation was found five nucleotides downstream from it (Fig.6A). These mutations caused a severe reduction in the level ofexpression of the icmF::lacZ fusion (data not shown).

From a comparison of the randomly mutated site in the

icmF regulatory region to the regulatory regions of icmV andicmW (these two genes are transcribed back to back and theirregulatory regions overlap) a 9-bp putative consensus sequence(CTATAGTAT) was identified (Fig. 6A). To determine theimportance of this site in the expression of icmV, icmW, andicmF, 2 nucleotides in each of these putative regulatory ele-ments were mutated (Fig. 6A). As can be seen in Fig. 6B, theeffect of these mutations on the icmW::lacZ and icmF::lacZfusions was dramatic. The expression of these lacZ fusions wasreduced about 10-fold when the fourth position of the consen-sus was mutated (mutation 2). In constructs, the effect on theicmV::lacZ fusion was minor, and only one of the mutationsresulted in a 20% reduction in expression. Because of thisresult and because icmV and icmW are transcribed back toback and the mutated sites overlap one another (Fig. 7; theregion is shown as double stranded), we decided to analyzetheir regulatory regions further, as described below.

icmV and icmW have overlapping regulatory regions. Care-ful analysis of the icmV and icmW overlapping regulatory re-gions revealed an interesting organization of several overlap-ping putative consensus sequences. The two sites examined inthe previous section (CTATAGTAT; one in the icmV regula-tory region and one in the icmW regulatory region; Fig. 7A)

FIG. 3. Analysis of the regulatory regions of icm::lacZ fusions that have low levels of expression. (A) Sequences of the regulatory regions oficmP, icmT, icmQ, and icmM are shown. The putative consensus sequence is in boldface, and the distances to the first ATG are indicated. Thenucleotides representing the 5� end of the mRNA are in boldface and underlined. Additional sequences that form potential inverted repeats withthe consensus found are boxed. Arrows 1 and 2, nucleotides mutated. The changes made were always A to C and T to G. (B) Expression oficmT::lacZ, icmP::lacZ, icmQ::lacZ, and icmM::lacZ fusions and of two mutant versions of each fusion in L. pneumophila at exponential phase wasdetermined. For each of the fusions, results for the wild-type (WT) regulatory region and for regulatory regions containing mutations in positions1 and 2 are shown. �-Galactosidase activity was measured as described in Materials and Methods. The data are the averages � standard deviations(error bars) of at least three different experiments. (C) Mapping of the 5� end of the icmP transcript by primer extension. Primer extension wasperformed as described in Materials and Methods with a primer complementary to the 5� end of the icmP gene. G, A, T, and C (top), productsof the sequencing reaction obtained by using the same primer. The sequence presented is that of the sense strand. Arrow, nucleotide representingthe 5� end of the mRNA. Boldface and underlining are as described for panel A.


overlap one another by 6 bp, and both of them also overlapanother possible regulatory element (Fig. 7A). In the icmWregulatory region, upstream from the CTATAGTAT site(overlapping by 2 nucleotides), there is a TATACT site (asequence identical to those of the �10 promoter elementsfound in icmT, icmP, icmQ, and icmM). Moreover, in the icmVregulatory region, downstream from the CTATAGTAT site(overlapping by three nucleotides), there is a TATATT site (asequence identical to part of the consensus sequence found inicmR). The organization of these sites in relation to one an-other is presented in Fig. 7A.

To determine the involvement of the sites mentioned abovein the expression of icmV and icmW, four additional mutationsin this region were constructed and their effect on the expres-sion of these fusions was determined (Fig. 7B). In icmW, mu-tations in both sites (CTATAGTAT and TATACT) result indramatic reduction in �-galactosidase activity. In icmV, themutations in the CTATAGTAT putative consensus had a mi-nor effect on the expression of the lacZ gene but the change inthe third position of the TATATT putative consensus reducedthe expression to less then 20%.

To determine which of these sites, if any, constitute the �10promoter elements of the icmV and icmW genes, the transcrip-tion start sites of both genes were determined, and the primerextension analysis of the icmV gene is presented in Fig. 7C.This analysis clearly indicates that TATACT in icmW and

TATATT in icmV constitute the �10 promoter elements ofthese genes. The 9-bp consensus sequence (CTATAGTAT)identified probably serves as a binding site for a transcriptionregulator.

DISCUSSION

L. pneumophila is an intracellular pathogen that inhibitsphagosome-lysosome fusion and that grows inside human mac-rophages and amoebae (28, 38). Thus far, 24 icm and dot genesrequired for intracellular growth have been found and charac-terized (44, 51). Even though these genes have been known forsome time, there is no information concerning DNA regulatoryelements and regulatory factors that control their expression.

It has been suggested that two major stationary-phase-re-lated factors (RpoS and RelA) are involved with L. pneumo-phila pathogenicity (4, 11, 21). However, the RpoS sigma fac-tor was shown to be dispensable for intracellular growth inHL-60-derived macrophages and THP-1 cells (20), and theRelA regulatory factor was shown to be dispensable for intra-cellular growth in both amoebae and HL-60-derived humanmacrophages (55). In contrast, the icm and dot genes wereshown to be required for intracellular growth in all the hostsexamined (HL-60- and U937-derived human macrophages [41,52], murine bone marrow-derived macrophages [50], and pro-tozoan hosts A. castellanii [45] and Dictyostelium discoideum

FIG. 4. Analysis of the regulatory region of the icmQ gene. (A) The sequence of the regulatory region of icmQ is shown (boldface, putativeconsensus sequence). The nucleotide representing the 5� end of the mRNA is in boldface and underlined. Arrows �1 to 7, nucleotides mutated.The changes made were always A to C, T to G, and C to A. (B) The expression of the icmQ::lacZ fusion (WT) and of the eight mutants from panelA in L. pneumophila at exponential phase was measured. �-Galactosidase activity was measured as described in Materials and Methods. The resultsare the averages � standard deviations (error bars) of at least three different experiments. (C) Mapping of the 5� end of the icmQ transcript byprimer extension. Primer extension was performed as described in Materials and Methods with a primer complementary to the 5� end of the icmQgene. G, A, T, and C (top), products of the sequencing reaction obtained by using the same primer. The sequence presented is that of the sensestrand. Arrow, nucleotide representing the 5� end of the mRNA. Boldface and underlining are as described for panel A.


[47]). In addition, analysis of the effect of a strain containing aninsertion in the rpoS gene (20) or the relA gene (55) on theexpression of the nine icm::lacZ fusions described revealedthat the expression of only one fusion (icmP::lacZ) was mod-erately reduced (55). This might explain the increase in �-ga-lactosidase activity observed with the icmP::lacZ fusion at sta-tionary phase (Fig. 1), but it cannot explain the results for theicmT::lacZ, icmR::lacZ, icmM::lacZ, and icmF::lacZ fusions, inwhich higher levels of �-galactosidase at stationary phase thanat exponential phase were observed as well (Fig. 1).

One approach to identify regulatory factors that control theexpression of a certain gene is to identify the DNA regulatoryelements of the gene of interest and then to find regulatoryfactors that interact with these sites. To gain information aboutthe DNA regulatory elements that control icm and dot geneexpression, we analyzed their upstream regulatory regions byextensive site-directed and random mutagenesis as well asprimer extension analysis. We were able to identify 12 DNAregulatory elements that are involved in the regulation of eighticm genes. Seven of these sites constitute the �10 promoterelements of the icm genes, and the other five sites (two in theicmR regulatory region and one each in the icmV, icmW, andicmF regulatory regions) probably serve as binding sites for

regulatory proteins. The sequences of all these regulatory el-ements are summarized in Fig. 8.

Four icm::lacZ fusions that had low levels of expression(icmT, icmP, icmQ, and icmM fusions) were found to containa 6-bp consensus sequence (TATACT) essential for their ex-pression (Fig. 3). As can be seen in Fig. 8, these four genes canbe divided into two pairs. In icmT and icmP there are fouradditional nucleotides (AGTA), located immediately up-stream from the TATACT regulatory element, that can forman inverted repeat that overlaps the regulatory element found.In addition, these two genes were found to be expressed atsimilar levels in L. pneumophila, and both of them had higherlevels of expression at stationary phase than at exponentialphase (Fig. 1). The second pair of genes is icmQ and icmM.These two genes have only 2 bp (GT) of identical sequenceimmediately upstream from the TATACT site. In addition,they contain two identical sequences of 6 and 5 nucleotides(AGTGAT and TATAA, respectively) located upstream fromthe TATACT site. One of these sequences (TATAA) can forma potential inverted repeat that overlaps the regulatory ele-ment (Fig. 8). In addition, the expression of these two genes atboth stationary and exponential phases was at least seven timeshigher in E. coli than in L. pneumophila (compare Fig. 1 and 2).

FIG. 5. Analysis of the regulatory region of the icmR gene. (A). The sequence of the regulatory region of icmR is shown, and the distance fromthe first ATG is indicated. The two putative consensus sequences are boxed, and the part of the consensus sequence (TATATT) similar to theconsensus sequences found in the icmT, icmP, icmQ, and icmM genes is also indicated. The nucleotide representing the 5� end of the mRNA isin boldface and underlined, and the �10 promoter element is in boldface. Arrows 1 to 6, nucleotides mutated. The changes made were always Ato C and T to G. (B) The expression of the icmR::lacZ fusion (WT) and of the six mutants from panel A in L. pneumophila at exponential phasewas measured. �-Galactosidase activity was measured as described in Materials and Methods. The data are the averages � standard deviations(error bars) of at least three different experiments. (C) Mapping of the 5� end of the icmR transcript by primer extension. Primer extension wasperformed as described in Materials and Methods with a primer complementary to the 5� end of the icmR gene. G, A, T, and C (top), productsof the sequencing reaction obtained by using the same primer. The sequence presented is that of the sense strand. Arrow, nucleotide representingthe 5� end of the mRNA. Boldface and underlining are as described for panel A.


The examination of a representative gene from each pairclearly indicated that the TATACT site constitutes the �10promoter elements of these genes (Fig. 3 and 4). The othersites described might serve as binding sites for regulatory pro-teins. For icmQ and icmM the sites indicated might be used asrepressor binding sites, a repressor which is probably missing inE. coli, and as a result of that the expression of the icmQ::lacZand icmM::lacZ fusions in E. coli increases.

icmJ is the only icm gene that was found to have low expres-sion levels, but the TATACT consensus was not found in itsregulatory region (even when a 300-bp regulatory region wascloned upstream of the lacZ gene, the same low level of ex-pression was observed [data not shown]). A potential site thathas 2 nucleotides different from the consensus (TATCTT) wasthe closest match found, but, when the second and third nu-cleotides of this site were mutated, no change in the level ofexpression of the icmJ::lacZ fusion in L. pneumophila or E. coliwas observed (data not shown).

icmR is the only gene that was found to have higher levels ofexpression in L. pneumophila than in E. coli. This gene wasfound to contain a �10 promoter element similar to those oficmT, icmP, icmM, and icmQ, as determined by primer exten-sion (Fig. 5). In addition, we identified two identical regulatory

elements (AAGATATATT) in the upstream region of thisgene (Fig. 8) and showed that they play a role in icmR expres-sion. These sites might constitute a binding site for an activatormissing in E. coli. The 10-bp consensus sequence that wasfound in the icmR regulatory region was not found in any ofthe other icm regulatory regions. In addition, its �10 promoterelement differs from all the other �10 promoter elementsidentified (the last nucleotide of the icmR promoter is G in-stead of T; Fig. 8). This result is in agreement with the obser-vation that the icmR::lacZ fusion is the only fusion more highlyexpressed in L. pneumophila than in E. coli. This might indicatethat the icmR gene is subjected to a unique regulation notpresent in the other icm genes.

By random mutagenesis and sequence alignment a 9-bp reg-ulatory element (CTATAGTAT) (Fig. 8) was identified in theupstream regions of the three genes (icmF, icmV, and icmW)that were found to have high expression levels in both E. coliand L. pneumophila. This site was found to have an effect onthe expression of these genes, but primer extension analysisindicated that it does not serve as a �10 promoter element.The �10 promoter elements that were determined by primerextension were found to be similar to the �10 promoter ele-ments of the other icm genes (Fig. 8). The �10 promoterelement and the regulatory elements identified in icmV andicmW were found to overlap one another in both genes andbetween the two genes (Fig. 7A). This organization mightindicate that these two genes are subjected to complicatedregulation.

As can be seen in Fig. 8, all the sites that were identified as�10 promoter elements have extensive homology to one an-other and are probably recognized by one of the L. pneumo-phila sigma factors. As most of the genome of L. pneumophilahas already been sequenced (http://genome3.cpmc.columbia.edu/legion/index.html), we looked for all the genes that havehomology to known sigma factors and found that L. pneumo-phila contains homologs to at least six sigma factors (RpoD,RpoH, RpoF, RpoE, RpoS, and RpoN). The promoter se-quence recognized by two of these sigma factors in L. pneu-mophila is already known (2, 22). RpoH and RpoF recognizesequences similar to the ones recognized by their E. coli ho-mologs (CTTGAAA[11 to 16]CCCATnT for RpoH [n A, G,T, or C] and TAAA[15]GCCGATAA for RpoF). These se-quences are different from the ones found in the icm genepromoter regions, which might indicate that these two sigmafactors are not involved in the recognition of the icm promot-ers. The sequence recognized by the L. pneumophila RpoE andRpoN sigma factors is not known. But it was shown that boththe E. coli RpoE and its Pseudomonas aeruginosa homologrecognize the same promoter sequence (16) (GAACTT[16 to17]TCTGA), and the sequence recognized by the RpoN sigmafactor is known for many bacteria (TGGCAC[5]TTGC) (6).Because all these bacteria are evolutionarily closely related toone another (they are all � purple bacteria), it is most likelythat these two sigma factors recognize similar promoter se-quences in L. pneumophila. However, as these sequences werenot found in the regulatory regions of the icm genes, we con-cluded that neither of these sigma factors is involved in therecognition of the icm promoters. Both the vegetative sigmafactor, RpoD, and the stationary-phase sigma factor, RpoS, ofE. coli recognize very similar �10 promoter elements (TAT

FIG. 6. Analysis of the regulatory region of icm::lacZ fusions thathave high expression levels. (A) Sequences of the regulatory regions oficmW, icmV, and icmF. The distances to the first ATG are indicated.The region of sequence similarity is boxed, and the sequence (TATAGT) similar to those of the �10 promoter elements of the icmT, icmP,icmQ, and icmM genes is indicated. �, nucleotides in the icmF regu-latory region in which random mutations were identified; arrows 1 and2, nucleotides mutated by site-directed mutagenesis. The changesmade were always A to C and T to G. (B) The expression oficmW::lacZ, icmV::lacZ, and icmF::lacZ fusions as well as of two mu-tant versions of each of these fusions in L. pneumophila at exponentialphase was measured. For each of the genes, the wild-type (WT) reg-ulatory region and the regulatory regions containing mutations inpositions 1 and 2 are shown. �-Galactosidase activity was measured asdescribed in Materials and Methods. The data are the averages �standard deviations (error bars) of at least three different experiments.


AAT for RpoD and CTATACT for RpoS) (53). This sequenceis also similar to the sequence of the �10 promoter elementthat we found in the icm gene regulatory regions (TATAYT).However, as we recently demonstrated (55), a knockout of therpoS gene moderately influenced the expression of only one(icmP::lacZ) out of the nine icm::lacZ fusions described. Tak-ing all this information together we believe that the �10 pro-moter element of the icm and dot genes is recognized by thevegetative sigma factor (RpoD) of L. pneumophila. Furthersupport for this assumption comes from the analysis of theicmQ �10 promoter element (Fig. 4). This analysis revealedthat the first 2 nucleotides and the last nucleotide of this �10promoter element are more important for expression then theother nucleotides. A similar situation was also found with theE. coli �10 promoter element of the vegetative sigma factor(31).

It is well known that promoters recognized by the vegetativesigma factor contain a conserved �35 promoter element inaddition to the conserved �10 promoter element (53). Exam-ination of the sequence located at the region where the �35promoter element should have been located revealed that noobvious consensus sequence can be found in this region (besidethe sequences found in the icmM and icmQ regulatory regions)

(Fig. 8). However, this is not the first case where a �10 pro-moter element is present and a �35 element is missing. Fortwo other bacteria, Helicobacter pylori (17) and Agrobacteriumtumefaciens (14), a similar situation, which also involve viru-lence-related genes, was described. In A. tumefaciens it isknown that a two-component regulatory system (virA and virG)is involved in the expression of the virulence-related genes(48).

However, two additional sites might be involved in the ex-pression of genes beside the �10 and �35 promoter elements.An extended �10 promoter element was described for severalsystems (7, 12, 30). The sequence of the extension is composedof 2 nucleotides (usually TG), located 1 bp upstream from the�10 promoter element, and this sequence was shown to inter-act with region 2.5 of the vegetative sigma factor (5). A se-quence identical to that of the known extended �10 promoterelement was found only in the icmR promoter (Fig. 8), but itmight be that in L. pneumophila other sequences constitute anextended �10 promoter that functions instead of a �35 pro-moter element in the icm genes. A second sequence that wasfound to be part of several promoters recognized by the veg-etative sigma factor is called the UP element (19, 37). Thissequence was found to be located upstream from the �35

FIG. 7. Analysis of the icmW and icmV regulatory regions. (A) The sequences of the regulatory regions of icmW and icmV are shown. Thesetwo genes are transcribed back to back, and the region is shown as double stranded. The 9-bp consensus sequence is boxed, and the internal 6-bpsequence (TATAGT) similar to the promoter elements found in the icmT, icmP, icmQ, and icmM genes is indicated. The nucleotides representingthe 5� end of the mRNA are in boldface and underlined, and the �10 promoter elements are in boldface. Arrows 1 to 4, nucleotides mutated. Thechanges made were always A to C and T to G. (B) The expression of icmW::lacZ, and icmV::lacZ fusions as well as four mutant versions of eachof these fusions in L. pneumophila at exponential phase was measured. For each of the genes, the wild-type (WT) regulatory region and theregulatory regions containing mutations in positions 1, 2, 3, and 4 are shown. �-Galactosidase activity was measured as described in Materials andMethods. The data are the averages of at least three different experiments. (C) Mapping of the 5� end of the icmV transcript by primer extension.Primer extension was performed as described in Materials and Methods with a primer complementary to the 5� end of the icmV gene. G, A, T,and C (top), products of the sequencing reaction obtained by using the same primer. The sequence presented is that of the sense strand. Arrow,nucleotide representing the 5� end of the mRNA. Boldface and underlining are as described for panel A.


promoter element in the region between nucleotides �59 and�42 with respect to the transcription start site, and it is anAT-rich sequence (AAA[A/T][A/T]T[A/T]TTTTNNAAAA)(15) that interacts with the alpha subunit of RNA polymerase(36). No sequence similar to the one presented was found inany of the icm promoter regions.

It is highly possible that additional regulatory elements, likethe sequences described, that form potential inverted repeatswith the �10 promoter element (Fig. 8) participate in theregulation of the icm genes. In addition, since we used trans-lational fusions (for each gene, the codons for the first sevenamino acids were included in the fusion) to determine theexpression levels of the different icm genes, we cannot rule outcontributions by posttranscriptional events to the differences inthe expression observed.

We believe that the icm and dot genes are subjected tocomplicated regulation, which is required for the correct andsuccessful function of their gene products. The fact that genesthat are part of the same virulence system are expressed atdifferent levels and contain similar as well as different DNAregulatory elements might indicate that a regulatory networkthat includes factors that respond to different environmentaland growth conditions participates in their regulation. We be-gan here to explore this regulatory network by finding 12 reg-ulatory elements involved in the regulation of eight icm and dotgenes and operons. The identification of these sites will allowus to find additional sites and regulatory factors that respondto changes in environmental signals and growth conditions andthat participate in the regulation of the icm/dot virulence ma-chinery.

ACKNOWLEDGMENTS

We are grateful to Howard A. Shuman for carefully reading themanuscript.

The research was supported by the Charles H. Revson Foundationof the Israel Science Foundation (grant 45/00). G. Segal was supportedby the Alon fellowship awarded by the Israeli Ministry of Education.

REFERENCES

1. Abu Kwaik, Y. 1996. The phagosome containing Legionella pneumophilawithin the protozoan Hartmannella vermiformis is surrounded by the roughendoplasmic reticulum. Appl. Environ. Microbiol. 62:2022–2028.

2. Amemura-Maekawa, J., and H. Watanabe. 1997. Cloning and sequencing ofthe dnaK and grpE genes of Legionella pneumophila. Gene 197:165–168.

3. Andrews, H. L., J. P. Vogel, and R. R. Isberg. 1998. Identification of linkedLegionella pneumophila genes essential for intracellular growth and evasionof the endocytic pathway. Infect. Immun. 66:950–958.

4. Bachman, M. A., and M. S. Swanson. 2001. RpoS co-operates with otherfactors to induce Legionella pneumophila virulence in the stationary phase.Mol. Microbiol. 40:1201–1214.

5. Barne, K. A., J. A. Bown, S. J. Busby, and S. D. Minchin. 1997. Region 2.5of the Escherichia coli RNA polymerase sigma70 subunit is responsible forthe recognition of the �extended-10�’ motif at promoters. EMBO J. 16:4034–4040.

6. Barrios, H., B. Valderrama, and E. Morett. 1999. Compilation and analysisof sigma(54)-dependent promoter sequences. Nucleic Acids Res. 27:4305–4313.

7. Bashyam, M. D., and A. K. Tyagi. 1998. Identification and analysis of “ex-tended �10” promoters from mycobacteria. J. Bacteriol. 180:2568–2573.

8. Berger, K. H., J. J. Merriam, and R. R. Isberg. 1994. Altered intracellulartargeting properties associated with mutations in the Legionella dotA gene.Mol. Microbiol. 14:809–822.

9. Bozue, J. A., and W. Johnson. 1996. Interaction of Legionella pneumophilawith Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibitionof phagosome-lysosome fusion. Infect. Immun. 64:668–673.

10. Brand, B. C., A. B. Sadosky, and H. A. Shuman. 1994. The Legionellapneumophila icm locus: a set of genes required for intracellular multiplica-tion in human macrophages. Mol. Microbiol. 14:797–808.

11. Byrne, B., and M. S. Swanson. 1998. Expression of Legionella pneumophilavirulence traits in response to growth conditions. Infect. Immun. 66:3029–3034.

12. Camacho, A., and M. Salas. 1999. Effect of mutations in the “extended-10”motif of three Bacillus subtilis sigmaA-RNA polymerase-dependent promot-ers. J. Mol. Biol. 286:683–693.

13. Casadaban, M. J., and S. N. Cohen. 1980. Analysis of gene control signals byDNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138:179–207.

14. Das, A., S. Stachel, P. Ebert, P. Allenza, A. Montoya, and E. Nester. 1986.Promoters of Agrobacterium tumefaciens Ti-plasmid virulence genes. NucleicAcids Res. 14:1355–1364.

15. Estrem, S. T., T. Gaal, W. Ross, and R. L. Gourse. 1998. Identification of anUP element consensus sequence for bacterial promoters. Proc. Natl. Acad.Sci. USA 95:9761–9766.

16. Firoved, A. M., J. C. Boucher, and V. Deretic. 2002. Global genomic analysisof AlgU �E-dependent promoters (sigmulon) in Pseudomonas aeruginosaand implications for inflammatory processes in cystic fibrosis. J. Bacteriol.184:1057–1064.

17. Forsyth, M. H., and T. L. Cover. 1999. Mutational analysis of the vacApromoter provides insight into gene transcription in Helicobacter pylori. J.Bacteriol. 181:2261–2266.

FIG. 8. Sequence alignment of the icm regulatory regions. The sequences are aligned according to the �10 promoter elements (boldface). Thetranscription start sites are in boldface and are underlined. �, mutations (29 in all). In icmP, icmT, icmQ, and icmM, sequence homologies betweenpairs of genes are boxed. In icmR, the 10-nucleotide direct repeat is boxed. In icmV, icmW, and icmF, the 9-nucleotide regulatory element is boxed.Arrows (indicating the center of symmetry), potential inverted repeats. The distance to the first ATG of each gene is indicated. CONC., consensus.


18. Fromant, M., S. Blanquet, and P. Plateau. 1995. Direct random mutagenesisof gene-sized DNA fragments using polymerase chain reaction. Anal. Bio-chem. 224:347–353.

19. Gourse, R. L., W. Ross, and T. Gaal. 2000. UPs and downs in bacterialtranscription initiation: the role of the alpha subunit of RNA polymerase inpromoter recognition. Mol. Microbiol. 37:687–695.

20. Hales, L. M., and H. A. Shuman. 1999. The Legionella pneumophila rpoSgene is required for growth within Acanthamoeba castellanii. J. Bacteriol.181:4879–4889.

21. Hammer, B. K., and M. S. Swanson. 1999. Co-ordination of Legionellapneumophila virulence with entry into stationary phase by ppGpp. Mol.Microbiol. 33:721–731.

22. Heuner, K., J. Hacker, and B. C. Brand. 1997. The alternative sigma factor�28 of Legionella pneumophila restores flagellation and motility to an Esch-erichia coli fliA mutant. J. Bacteriol. 179:17–23.

23. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989.Site-directed mutagenesis by overlap extension using the polymerase chainreaction. Gene 77:51–59.

24. Horwitz, M. A. 1983. Formation of a novel phagosome by the Legionnaires’disease bacterium (Legionella pneumophila) in human monocytes. J. Exp.Med. 158:1319–1331.

25. Horwitz, M. A. 1983. The Legionnaires’ disease bacterium (Legionella pneu-mophila) inhibits phagosome-lysosome fusion in human monocytes. J. Exp.Med. 158:2108–2126.

26. Horwitz, M. A. 1984. Phagocytosis of the Legionnaires’ disease bacterium(Legionella pneumophila) occurs by a novel mechanism: engulfment within apseudopod coil. Cell. 36:27–33.

27. Horwitz, M. A., and F. R. Maxfield. 1984. Legionella pneumophila inhibitsacidification of its phagosome in human monocytes. J. Cell Biol. 99:1936–1943.

28. Horwitz, M. A., and S. C. Silverstein. 1980. Legionnaires’ disease bacterium(Legionella pneumophila) multiplies intracellularly in human monocytes.J. Clin. Investig. 60:441–450.

29. Komano, T., T. Yoshida, K. Narahara, and N. Furuya. 2000. The transferregion of IncI1 plasmid R64: similarities between R64 tra and Legionellaicm/dot genes. Mol. Microbiol. 35:1348–1359.

30. Kumar, A., R. A. Malloch, N. Fujita, D. A. Smillie, A. Ishihama, and R. S.Hayward. 1993. The minus 35-recognition region of Escherichia coli sigma 70is inessential for initiation of transcription at an “extended minus 10” pro-moter. J. Mol. Biol. 232:406–418.

31. Lisser, S., and H. Margalit. 1993. Compilation of E. coli mRNA promotersequences. Nucleic Acids Res. 21:1507–1516.

32. Marra, A., S. J. Blander, M. A. Horwitz, and H. A. Shuman. 1992. Identifi-cation of a Legionella pneumophila locus required for intracellular multipli-cation in human macrophages. Proc. Natl. Acad. Sci. USA 89:9607–9611.

33. Miller, J. H. 1972. Experiments in molecular biology. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

34. Nagai, H., J. C. Kagan, X. Zhu, R. A. Kahn, and C. R. Roy. 2002. A bacterialguanine nucleotide exchange factor activates ARF on Legionella phago-somes. Science 295:679–682.

35. Purcell, M. W., and H. A. Shuman. 1998. The Legionella pneumophilaicmGCDJBF genes are required for killing of human macrophages. Infect.Immun. 66:2245–2255.

36. Ross, W., A. Ernst, and R. L. Gourse. 2001. Fine structure of E. coli RNApolymerase-promoter interactions: alpha subunit binding to the UP elementminor groove. Genes Dev. 15:491–506.

37. Ross, W., K. K. Gosink, J. Salomon, K. Igarashi, C. Zou, A. Ishihama, K.Severinov, and R. L. Gourse. 1993. A third recognition element in bacterialpromoters: DNA binding by the alpha subunit of RNA polymerase. Science262:1407–1413.

38. Rowbotham, T. J. 1980. Preliminary report on the pathogenicity of Legionellapneumophila for freshwater and soil amoebae. J. Clin. Pathol. 33:1179–1183.

39. Sadosky, A. B., L. A. Wiater, and H. A. Shuman. 1993. Identification ofLegionella pneumophila genes required for growth within and killing ofhuman macrophages. Infect. Immun. 61:5361–5373.

40. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing withchain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.

41. Segal, G., M. Purcell, and H. A. Shuman. 1998. Host cell killing and bacterialconjugation require overlapping sets of genes within a 22-kb region of theLegionella pneumophila genome. Proc. Natl. Acad. Sci. USA 95:1669–1674.

42. Segal, G., and E. Z. Ron. 1993. Heat shock transcription of the groESLoperon of Agrobacterium tumefaciens may involve a hairpin-loop structure. J.Bacteriol. 175:3083–3088.

43. Segal, G., and H. A. Shuman. 1997. Characterization of a new region re-quired for macrophage killing by Legionella pneumophila. Infect. Immun.65:5057–5066.

44. Segal, G., and H. A. Shuman. 1998. How is the intracellular fate of theLegionella pneumophila phagosome determined? Trends Microbiol. 6:253–255.

45. Segal, G., and H. A. Shuman. 1999. Legionella pneumophila utilize the samegenes to multiply within Acanthamoeba castellanii and human macrophages.Infect. Immun. 67:2117–2124.

46. Segal, G., and H. A. Shuman. 1999. Possible origin of the Legionella pneu-mophila virulence genes and their relation to Coxiella burnetii. Mol. Micro-biol. 33:669–670.

47. Solomon, J. M., A. Rupper, J. A. Cardelli, and R. R. Isberg. 2000. Intracel-lular growth of Legionella pneumophila in Dictyostelium discoideum, a systemfor genetic analysis of host-pathogen interactions. Infect. Immun. 68:2939–2947.

48. Stachel, S. E., and P. C. Zambryski. 1986. virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens. Cell46:325–333.

49. Swanson, M. S., and R. R. Isberg. 1995. Association of Legionella pneumo-phila with the macrophage endoplasmic reticulum. Infect. Immun. 63:3609–3620.

50. Vogel, J. P., H. L. Andrews, S. K. Wong, and R. R. Isberg. 1998. Conjugativetransfer by the virulence system of Legionella pneumophila. Science 279:873–876.

51. Vogel, J. P., and R. R. Isberg. 1999. Cell biology of Legionella pneumophila.Curr. Opin. Microbiol. 2:30–34.

52. Wiater, L. A., K. Dunn, F. R. Maxfield, and H. A. Shuman. 1998. Early eventsin phagosome establishment are required for intracellular survival of Legio-nella pneumophila. Infect. Immun. 66:4450–4460.

53. Wosten, M. M. 1998. Eubacterial sigma-factors. FEMS Microbiol. Rev. 22:127–150.

54. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phagecloning vectors and host strains: nucleotide sequences of the M13mp18 andpUC19 vectors. Gene 33:103–119.

55. Zusman, T., O. Gal-Mor, and G. Segal. 2002. Characterization of a Legio-nella pneumophila relA insertion mutant and the roles of RelA and RpoS invirulence gene expression. J. Bacteriol. 184:67–75.


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