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Infection and Immunity, April 2005, p. 2020-2032, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2020-2032.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Legionella pneumophila tatB Gene Facilitates Secretion of Phospholipase C, Growth under Iron-Limiting Conditions, and Intracellular Infection

Ombeline Rossier and Nicholas P. Cianciotto*

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois

Received 9 February 2004/ Returned for modification 10 March 2004/ Accepted 22 December 2004


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ABSTRACT
 
Our previous mutational analysis of Legionella pneumophila demonstrated a role for type II protein (Lsp) secretion and iron acquisition in intracellular infection and virulence. In gram-negative bacteria, the twin-arginine translocation (Tat) pathway is involved in secretion of proteins, including components of respiratory complexes, across the inner membrane to the periplasm. To assess the significance of Tat for L. pneumophila, tatB mutants were characterized. The mutants exhibited normal growth in standard media but grew slowly under low-iron conditions. They were also impaired in the Nadi assay, indicating that the function of cytochrome c oxidase is influenced by tatB. Consistent with this observation, a subunit of the cytochrome c reductase was shown to be a Tat substrate. Supernatants of the tatB mutants showed a 30% reduction in phospholipase C activity while maintaining normal levels of other Lsp secreted activities. When tested for infection of U937 macrophages, the tatB mutants showed a 10-fold reduction in growth. Double mutants lacking tatB and Lsp secretion were even more defective, suggesting tatB has an intracellular role that is independent of Lsp. tatB mutants were also impaired 20-fold in Hartmannella vermiformis amoebae cultured in the presence of an iron chelator. All mutant phenotypes were complemented by reintroduction of an intact tatB. Thus, L. pneumophila tatB plays a role in the formation of a respiratory complex, growth under low-iron conditions, the secretion of a phospholipase C activity, and intracellular infection.


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INTRODUCTION
 
The gram-negative bacterium Legionella pneumophila is the causal agent of Legionnaires' disease, a potentially fatal pneumonia that especially affects immunocompromised individuals (24, 73). A facultative intracellular parasite, it is ubiquitous in freshwater environments, where it replicates in biofilms and within protozoan hosts such as amoebae and ciliates (23, 73). Following aerosol inoculation of L. pneumophila into the human respiratory tract, bacterial multiplication occurs in alveolar macrophages and is accompanied by bacterial spread, host cell death, and damage to the lung tissue (73, 79).

We have been focused on the characterization of L. pneumophila type II protein secretion (2-4, 27, 39, 41, 62, 63). Present within multiple gram-negative bacteria, type II protein secretion allows protein export from the periplasm to the exterior milieu by a complex apparatus comprising ca. 12 proteins (25, 52, 64, 66). Secreted enzymatic activities or proteins dependent upon the Legionella secretion pathway (i.e., Lsp) include acid phosphatases, a zinc metalloprotease, an RNase, lipases, a phospholipase A (PLA), a lysophospholipase A (LPLA), and PLC (2-4, 26, 27, 31, 39, 62, 63). Importantly, L. pneumophila type II secretion mutants are defective for intracellular replication in amoebae and human macrophages (31, 39, 54, 62, 63). Furthermore, type II protein secretion is critical for multiplication and/or survival of L. pneumophila in the lungs of A/J mice following intratracheal infection (63). As has been seen with all other gram-negative bacteria studied, genetic determinants associated with L. pneumophila type II secretion include genes encoding an outer membrane secretin (lspD), an ATPase (lspE), a conserved inner membrane protein (lspF), pseudopilins (lspGHIJK), and a prepilin peptidase (pilD) (3, 31, 39, 62, 63).

Recently, studies in Pseudomonas aeruginosa have uncovered another type of gene that influences type II protein secretion, i.e., the twin-arginine translocation (tat) genes (46, 77). In gram-negative bacteria, secretion of proteins across the inner membrane occurs mainly by two pathways, i.e., the general secretion pathway (Sec) (57) and the Tat system (50). Unlike the Sec system, the Tat machinery is competent for the transport of fully folded proteins (50, 61) and, based upon the Escherichia coli model, includes the integral membrane proteins TatA, TatB, and TatC (10, 67, 78, 80). Signal peptides of Tat-dependently exported proteins resemble Sec-dependent signal peptides in their overall structure but differ in four characteristics from their Sec counterparts. First, they possess a so-called twin-arginine consensus motif (RRX{Phi}{Phi}, where {Phi} is a hydrophobic residue). Second, the consensus motif is followed by a hydrophobic region that is markedly less hydrophobic than that of Sec signal peptides. Third, basic residues are often found just before the signal peptidase cleavage sequence and could serve as a Sec avoidance signal. Fourth, Tat signal peptides can be much longer than the Sec signal sequences (50, 61). Although many of the known Tat substrates are periplasmic enzymes that bind redox cofactors (7, 10, 32, 43, 67, 78), two PLCs of P. aeruginosa that are secreted by the type II secretion system require the Tat machinery to travel across the inner membrane (46, 77). Thus, the Tat pathway has been linked to the secretion of virulence factors.

Therefore, based upon our long-standing interest in both L. pneumophila type II secretion and virulence, we were interested in the possible significance of an L. pneumophila Tat system. In addition to our interest in the Tat system for its possible role in L. pneumophila type II secretion, we pursued it for its possible connection to L. pneumophila iron acquisition, another focus of research in our laboratory. In P. aeruginosa, the Tat system is associated with siderophore production (46), and we have recently discovered an L. pneumophila siderophore (40). Furthermore, our mutational analysis has shown that genes such as feoB, iraAB, and ccm, which are important for extracellular growth under iron-limiting conditions, can also be determinants of intracellular growth and virulence (59, 75, 76). In this paper, we identified a role for the L. pneumophila tatB gene in secretion of PLC activity, growth under iron-limiting conditions, and intracellular infection.

(Portions of this work were presented at the 104th General Meeting of the American Society for Microbiology [O. Rossier and N. P. Cianciotto, Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr. B-150, 2004].)


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MATERIALS AND METHODS
 
Bacterial strains, media, and chemicals. L. pneumophila serogroup 1 strain 130b (ATCC BAA-74, and also known as AA100), which served as the wild type in this study, and its derivatives NU275 and NU257 that contain stable insertions of a kanamycin resistance (Kmr) gene in lspF and ccmC, respectively, were described previously (22, 63, 76). Strain GT282 (29), another derivative of 130b, carries a mini-Tn10 transposon in the ccmF gene and was a gift from Y. Abu Kwaik. Legionellae were routinely cultured at 37°C in buffered yeast extract (BYE) broth or on buffered charcoal yeast extract (BCYE) agar (20). Growth in liquid medium was assessed by measuring the optical density of the culture at 660 nm over 24 h (DU520 spectrophotometer; Beckman, Fullerton, Calif.). To assess bacterial growth under low-iron conditions, the legionellae were plated on BCYE agar that lacked its usual ferric pyrophosphate supplement (BCYE-Fe) and on BCYE-Fe agar supplemented with 0.4 mM of the ferrous iron chelator 2,2'-dipyridyl (DIP). In order to examine L. pneumophila siderophore production, bacteria were grown in iron-deplete chemically defined medium (40, 58). To further limit the amount of iron in the chemically defined medium cultures, deferrated Milli-Q water and acid-washed glassware were used (14). E. coli strains NovaBlue (Novagen, Madison, Wis.) and DH5{alpha} (Invitrogen, Carlsbad, Calif.), hosts for recombinant plasmids, as well as strains MC4100 (68) and its tatB mutant derivative BØD (68), were grown at 37°C on Luria-Bertani agar (5). Antibiotics were added to the media at the following final concentrations (in micrograms per milliliter): ampicillin, 100; chloramphenicol, 6 for L. pneumophila and 30 for E. coli; gentamicin, 2.5; and kanamycin, 25 for L. pneumophila and 50 for E. coli. Unless otherwise noted, chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo.).

DNA isolation, PCR, and sequence analysis. Genomic DNA was isolated from L. pneumophila as described previously (21). Based on data from the L. pneumophila Philadelphia-I genome database (http://genome3.cpmc.columbia.edu /legion/), primers were designed for amplifying genes from 130b DNA. Primers OR66tatB (5'-TACATGTGGAGTCAGGATGG) and OR67tatB (5'-CACGGCGTTCATAATCACAG) yielded a 1,732-bp fragment containing tatA and tatB. Sequencing reactions were performed using two different PCR amplicons, a series of custom primers, and the BigDye terminator cycle-sequencing mix (PE Applied Biosystems, Foster City, Calif.). Automated sequence analysis was done on an ABI Prism 373 DNA sequencer (Applied Biosystems) at the Biotech Facility at Northwestern University. Primers were obtained from Integrated DNA Technologies (Coralville, Iowa). A screen for proteins containing a twin-arginine motif encoded in the genomes of L. pneumophila strains Philadelphia-1 (12) and Paris (11) was performed using the pattern searches in the PEDANT website (http://pedant.gsf.de) (28) and the LegioList website (http://genolist.pasteur.fr/LegioList/genome.cgi), respectively. Other database searches were performed using programs based on the BLAST algorithm (1), and protein sequences were analyzed for signal peptides using the PSORT and SignalP programs (http://psort.nibb.ac.jp/ and http://www.cbs.dtu.dk/services/SignalP/) (44, 45).

Gene cloning, mutant generation, and complementation analysis. To facilitate construction of L. pneumophila mutants, the PCR fragment amplified with OR66tatB and OR67tatB was ligated into pGem-T Easy (Promega, Madison, Wis.), yielding pGtatAB. Plasmid pGtatAB was digested with HindIII, which cuts 61 bp after the tatB start codon and then, following Klenow treatment, was ligated either to a Kmr gene isolated from pMB2190 upon HincII digestion (30) to yield pGtatB::Km or to a gentamicin resistance (Gmr) gene isolated from pX1918GT after HincII and PvuII digestion (69) to give pGtatB::Gm. Finally, tatB mutants were obtained by natural transformation (27, 72) of strain 130b with pGtatB::Km and pGtatB::Gm. Verification of all mutant genotypes was carried out by PCR and Southern hybridization (data not shown). To construct a tatB lspF double mutant, a Gmr tatB mutant was transformed with pGlspF::Km, a plasmid containing a Kmr insertion in the L. pneumophila lspF gene (63). To construct a tatB pilD double mutant, a Kmr tatB mutant was transformed with pGD::Gm, a plasmid containing a Gmr insertion in the L. pneumophila pilD gene (63). To express the L. pneumophila tatB gene in E. coli and facilitate complementation of the tatB mutants in L. pneumophila, a 400-bp fragment containing only tatB was amplified by PCR from L. pneumophila Philadelphia-I genomic DNA using primers OR75tatB (5'-CACAGGTCTTGTCCGAGTTA) and OR76tatA (5'-CTGACCTTGGTGAAGCGATT) and then subcloned into pGem-T Easy. Following an EcoRI digestion of the resulting plasmid, the tatB gene was cloned under the control of the tac promoter in pMMB2002 (63), yielding pMtatB. Plasmids were electroporated into L. pneumophila strains as previously described (13).

Microscopic analysis and sensitivity assays. Light microscopic analysis of E. coli and L. pneumophila broth cultures (n = 2) was performed on wet mounts by using a Labophot phase-contrast microscope (Nikon, Melville, N.Y.) with a 40x objective and a 100x oil immersion objective. Sensitivities of L. pneumophila strains to sodium dodecyl sulfate (SDS), rifampin, erythromycin, and DIP were tested using disk diffusion (19) and microdilution assays (60). For the disk diffusion assay, bacterial strains were grown at 37°C on BCYE agar for 3 days or in BYE broth to stationary phase and then resuspended in BYE to 5 x 106 CFU/ml. After 100 µl of the bacterial suspension was spread on BCYE agar using a sterile cotton swab, 6-mm disks containing SDS (1 mg), rifampin (340 µg), erythromycin (15 µg), or DIP (781 µg) were placed on the agar plates (n = 2 to 3). The diameters of the zones of growth inhibition were measured after the plates were incubated for 40 h at 37°C (19). For the microdilution assay, bacterial strains were grown at 37°C on BCYE agar for 3 days or in BYE broth to stationary phase and then resuspended in BYE to 5 x 104 CFU/ml. In triplicate wells of a 96-well microtiter polystyrene tray, 50 µl of the bacterial suspension was added to an equal volume of BYE containing various concentrations of SDS, rifampin, erythromycin, or DIP. Following incubation at 37°C for 40 h, the lowest concentration of detergent, antibiotic, or iron chelator resulting in no visible growth was deemed to be the MIC for that compound (60).

Determination of cytochrome c-dependent respiration. The classical Nadi assay was used to detect the aerobic respiratory capacity of the cytochrome c oxidase branch (42, 43, 49). L. pneumophila colonies grown for 3 days at 37°C on BCYE agar were submerged for 15 s with a freshly prepared 1:1 mixture of 1% {alpha}-naphtol in 95% ethanol and 1% N,N-dimethyl-p-phenylenediamine monohydrochloride in water, and then the plate was drained and exposed to air. In a positive reaction colonies stain a deep-blue color, whereas Nadi-negative colonies remain white (42, 43, 49).

Analysis of secreted activities. Filter-sterilized supernatants from L. pneumophila cultures in BYE broth in late exponential phase (3) were tested for PLC activity as determined by the release of p-nitrophenol (p-NP) from p-nitrophenol phosphorylcholine (p-NPPC) (3, 4, 6, 46, 77). Protease activity was determined using azocasein as a substrate as previously described (3). Tartrate-sensitive and tartrate-resistant acid phosphatases were monitored by the release of p-NP from p-NP phosphate in 200 mM sodium acetate, pH 5.5, in the absence or presence of 5 mM tartrate (2, 3). RNase activity was assayed by monitoring the release of soluble nucleotides from Baker's yeast type III RNA, as previously described (63). Monoacylglycerol lipase, PLA, and LPLA activities were measured by the ability of supernatants to release free fatty acids from 1-monopalmitoyl glycerol, phosphatidylcholine, and lysophosphatidylcholine, respectively (3, 27, 62). Lipolytic activities were also determined by p-NP palmitate and p-NP caprylate hydrolysis (3, 4). To examine L. pneumophila siderophore production, culture supernatants were tested for reactivity in the chrome azurol S (CAS) assay (40, 51).

Cloning, epitope tagging, and immunodetection of L. pneumophila proteins. To facilitate immunodetection, L. pneumophila proteins PetA, CcmE, and PlcA were tagged with the FLAG epitope (DYKDDDDK) at their C-terminal ends. Three pairs of primers were designed for amplifying the corresponding genes from 130b DNA. Primers OR101FeSBglII (5'-TAATCCAGATCTTGCATCCTCACCTATCA) and OR96FeSEcoRI (5'-TTGTGAATTCTCTAAGAAATAAGTTATCCA) yielded a 759-bp fragment containing petA, OR93CcmEEcoRI (5'-GCATGAATTCTTTAGGAATAAAGTGGCAA) and OR100CcmEBglII (5'-TCCGAGATCTTTGTTTCACCTTATCAGC) yielded a 518-bp fragment encoding ccmE, and OR99PlcAKpnI (5'-TCTCACCGGTACCAATGATCAATCCCTGTT) and OR98PlcASalI (5'-TCTGCTCGTCGACCAAAGGATTATCTTGCAAT) yielded a 1,321-bp fragment containing plcA (restriction sites are underlined). The PCR fragments were ligated into pUC119 linearized with HincII (74), generating pUpetA, pUccmE, and pUplcA. Following digestion of pUpetA and pUccmE with EcoRI and BglII, petA and ccmE were cloned into pFLAG-CTC (Sigma-Aldrich), yielding ppetA-FLAG CTC and pccmE-FLAG CTC, respectively. Following digestion of plasmid pUplcA with KpnI and SalI, plcA was cloned into pFLAG-CTC to give pplcA-FLAG CTC. Next, ppetA-FLAG CTC and pccmE-FLAG CTC were digested with EcoRI and XmnI to clone petA-FLAG and ccmE-FLAG under the control of the tac promoter in pMMB2002 (63), yielding pMpetA and pMccmE, respectively. Similarly, plasmid pplcA-FLAG CTC was digested with KpnI and XmnI to clone plcA-FLAG into pMMB2002, resulting in plasmid pMplcA.

To express the fusion proteins in E. coli, bacteria were grown in Luria-Bertani broth to mid-exponential phase and subsequently grown with 2 mM isopropyl ß-D-thiogalactopyranoside (IPTG) for 2 h. For the production of the epitope-tagged proteins in L. pneumophila, bacteria were grown in BYE broth to late exponential phase and subsequently grown with 0.5 mM IPTG for 3 h. Bacteria were harvested by centrifugation for 2 min at 10,000 x g and resuspended in a 1:700 volume of Laemmli buffer (36). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 12% acrylamide for PetA-FLAG and CcmE-FLAG and 10% acrylamide for PlcA-FLAG) (65) and transferred to a nitrocellulose membrane using the Trans-Blot SD semidry electrophoretic transfer cell according to the manufacturer's instructions (Bio-Rad, Hercules, Calif.). The membrane was blocked for 30 min with 3% skim milk diluted in 10 mM Tris-HCl, pH 8.0, containing 150 mM NaCl and 0.05% polyoxyethylenesorbitan monolaureate (TBST) at room temperature, incubated overnight with the anti-FLAG M2 monoclonal antibody (Sigma-Aldrich; 1:1,000 dilution in TBST with 1% skim milk), washed three times for 10 min in TBST at room temperature, and incubated for 1 h with an anti-mouse immunoglobulin G peroxidase-conjugated antibody (Chemicon International, Temecula, Calif.; 1:5,000 dilution in TBST with 1% skim milk). After four washes with TBST, immunodetection was performed using the enhanced chemiluminescence Western blotting reagents (Amersham Biosciences, Piscataway, N.J.) and a charge-coupled device camera (Chemi Imager 5500; Alpha Innotech, San Leandro, Calif.).

Intracellular infection of protozoa and macrophages. To examine the ability of L. pneumophila to grow within a protozoan host, Hartmannella vermiformis was infected as previously described (2, 3, 13, 59, 62, 63). Thus, ca. 104 CFU was added to wells containing 105 amoebae and then, at various times postinoculation, the numbers of bacteria per coculture were determined by plating serial dilutions on BCYE agar supplemented with the appropriate antibiotics. To quantitate intracellular growth in human macrophages, U937 cells, a human monocyte cell line, were differentiated into macrophage-like cells by treatment with phorbol myristate acetate and were infected as previously described (2, 3, 39, 53, 59, 62, 63). Briefly, monolayers containing 106 macrophages were inoculated with approximately 105 CFU, incubated for 2 h to allow bacterial entry, and then washed three times with medium to remove unincorporated bacteria. At various times postinoculation, serial dilutions of the lysed monolayers were plated on BCYE agar, supplemented with antibiotics when appropriate, to determine the numbers of bacteria per monolayer. Infection of iron-depleted macrophages and amoebae was accomplished by the addition of various concentrations of DIP to the medium 24 h prior to infection with L. pneumophila and during the incubation period (59). All stages of the infections, including washes, were carried out in the appropriate concentrations of DIP. Infections of macrophages in the presence of additional iron were performed by supplementing the medium with 0.5 mM ferrous sulfate throughout the experiment.

Nucleotide sequence accession number. The nucleotide sequence of L. pneumophila strain 130b tatAB was deposited in GenBank under accession number AY588131.


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RESULTS
 
Identification, sequence, and cloning of L. pneumophila tatB. The incomplete genome database of L. pneumophila strain Philadelphia-1 (http://genome3.cpmc.columbia.edu/~legion/) was examined for genes encoding proteins homologous to E. coli TatA, TatB, and TatC (10, 67, 78). Two adjacent genes (tatAB) whose protein products appeared to have homology to TatA and TatB were identified. Immediately upstream of tatA were three genes, two of which (i.e., ubiE and ubiB) are predicted to play a role in ubiquinone synthesis (38, 55), and downstream of tatB there was an open reading frame that is transcribed in the opposite direction and whose predicted protein does not bear homology to any proteins in the GenBank database. The tatAB genes and their flanking regions were cloned from our virulent serogroup 1 strain 130b. Complete sequence analysis of the cloned genes confirmed that the L. pneumophila 6.5-kDa TatA is 74% similar to E. coli TatA (accession number O65938) and the L. pneumophila 10-kDa TatB bears 52% similarity to E. coli TatB (accession number O69415). Like their counterparts (33), L. pneumophila TatA and TatB were predicted to bear a transmembrane domain in their N terminus followed by an amphipathic helix. In an unlinked region of the genome, an open reading frame encoding a protein with 62% similarity to E. coli TatC (accession number P27857) was identified. Since the completion of this study, the completed genome sequences of L. pneumophila strains Philadelphia-1, Paris, and Lens have been published (11, 12). These reports confirmed the presence of genes encoding TatA, TatB, and TatC in L. pneumophila.

As additional confirmation of the identity of the Legionella tat genes, we examined the ability of cloned L. pneumophila tatB to function in E. coli in a manner analogous to that previously described for cloned E. coli tatB (68). Toward that end, the tatB gene from strain 130b was cloned under the control of the tac promoter in plasmid pMMB2002, and then the resulting plasmid, pMtatB, was introduced into E. coli strain MC4100 and its tatB mutant derivative BØD (68). E. coli tat mutants exhibit a filamentous phenotype (71), due to the mislocalization of cell wall-modifying amidases that are necessary for cell septation (8, 34). Moreover, the overexpression of tatB in wild-type E. coli inhibits the Tat system, leading to a tat-negative filamentous phenotype (68). Therefore, we tested whether overexpression of the L. pneumophila tatB gene in strain MC4100 would have a comparable effect. In the absence of IPTG induction, MC4100(pMtatB) showed normal septation, i.e., only 0.2% of the cells formed filaments (Table 1). In contrast, in the presence of 2 mM IPTG, 56% of the bacteria were filamentous, a level of filamentation that was comparable to that of the E. coli BØD tatB mutant (Table 1). Importantly, when the expression of two other L. pneumophila genes, i.e., petA and ccmE (see below), were similarly induced with IPTG in strain MC4100, the bacteria did not exhibit the septation defect (Table 1), indicating that not all L. pneumophila proteins inhibit E. coli septation. These results indicate that, similarly to E. coli TatB, L. pneumophila TatB can exhibit a dominant negative effect on the Tat system. In E. coli, to observe complementation of the tatB deletion in strain BØD the trans expression of the wild-type gene had to be modulated by decreasing the plasmid copy number (68). Therefore, we assessed the effect of introducing the L. pneumophila tatB gene in strain BØD in the absence of IPTG. Under this circumstance, strain BØD(pMtatB) showed a 50% decrease in filamentation compared to BØD(pMMB2002) (Table 1), indicating that the E. coli tatB mutation can be partially complemented by the L. pneumophila tatB gene. Taken together, these observations indicate that L. pneumophila tatB is functioning as expected for a gene involved in twin-arginine translocation.


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  		TABLE 1. Filamentous phenotypes of E. coli strains expressing L. pneumophila proteins

Mutation of L. pneumophila tatB and its effect on general growth characteristics. In order to assess the role of TatB in L. pneumophila, we used allelic exchange to isolate 130b mutants containing an antibiotic resistance gene inserted into tatB. One Kmr tatB mutant (i.e., NU287) and one Gmr tatB mutant (i.e., NU288) were generated. To evaluate the importance of tatB for L. pneumophila extracellular growth, we compared, on five different occasions, strains 130b, NU287, and NU288 for their growth at 37°C in BYE broth, the standard medium for culturing legionellae. As measured by the optical density at 660 nm of the cultures, the tatB mutants grew similarly to wild type throughout the exponential and stationary phases of growth (data not shown). Moreover, following 3 days of incubation on BCYE agar, the mutants gave colonies of similar size to those of strain 130b (data not shown). These data indicate that L. pneumophila tatB is not required for normal extracellular growth. During exponential and stationary phases in BYE broth, NU287 and NU288 exhibited a cell filamentation pattern typical for the wild type (data not shown), indicating that tatB is not required for normal septation in L. pneumophila as it is in E. coli. Since E. coli tat mutants show increased sensitivity to SDS and certain antibiotics (34, 71), we tested the susceptibility of the L. pneumophila tatB mutants to SDS, rifampin, and erythromycin. In a disk diffusion assay on BCYE agar, comparable growth inhibition zones were observed for the parental strain and its tatB derivatives (data not shown). Accordingly, the MICs of SDS, rifampin, and erythromycin for bacteria growing in BYE broth were identical for 130b, NU287, and NU288 (data not shown), suggesting that the L. pneumophila tatB mutants do not have major envelope defects.

Role of L. pneumophila tatB in cytochrome c-dependent respiration. A bacterial function that is commonly associated with the Tat system is the formation of respiratory complexes, such as those involved in cytochrome c-dependent respiration (43). Therefore, we next tested the effect of the tatB mutation on L. pneumophila cytochrome c-dependent respiration by using the classical Nadi assay (42, 43). In this assay, {alpha}-naphtol and dimethyl-p-phenylenediamine are oxidatively converted to the blue dye indophenol blue through the activity of cytochrome c and cytochrome c oxidase, i.e., Nadi-positive colonies appear blue. Indeed, whereas wild-type L. pneumophila 130b appeared blue on BCYE agar within 10 min of applying the Nadi reagents, two cytochrome c maturation (ccm) mutants of strain 130b remained white for at least 1 h. As we predicted, the tatB mutants NU287 and NU288 were also negative in the Nadi assay. Incidentally, an L. pneumophila lspF mutant was not defective, indicating that type II secretion is not needed for cytochrome c-dependent respiration. Introduction of tatB-containing pMtatB into NU287 and NU288 restored the positive Nadi reaction. These data indicate that tatB is needed for cytochrome c-dependent respiration in L. pneumophila.

Influence of tatB on processing of the cytochrome c reductase PetA. In order to identify Tat substrates that are associated with Legionella cytochrome c-dependent respiration and that might also serve as markers to document Tat processing in L. pneumophila, we used the PEDANT and LegioList websites to screen the genome sequences of L. pneumophila strains Philadelphia-1 and Paris for proteins containing two consecutive arginines in their N terminus. Additional criteria used were the presence of a hydrophobic region of amino acids downstream of the RR motif and the identification of a signal peptide by PSORT or SignalP programs. Table 2 lists the putative Tat substrates identified in this screen. One protein that met our criteria was PetA, a protein homologous to the Rieske iron-sulfur subunit of ubiquinol-cytochrome c reductases of Vibrio vulnificus and others (accession number NP759586) (48). This L. pneumophila protein, like some known Tat substrates in other bacteria, exhibited an unusually long N-terminal signal sequence, i.e., MSEMTDLNQDVSNHNQDEQLDEERRRFLLHTTCVLSGVGAACALTPFITSWLPSSKAQA. When we applied the recently described TATFIND 1.2 algorithm (17), PetA was again identified as a potential Tat substrate. Another protein identified in the screen was CcmE, a periplasmic chaperone for cytochrome c maturation that we previously described (76). The N terminus of CcmE is MTPVRRRKLFILLFALSVLSAAA.


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  		TABLE 2. Potential Tat substrates in L. pneumophila strainsa

Since PetA and CcmE were identified as potential substrates of the Tat system in L. pneumophila, we investigated whether their signal peptide could be cleaved in a tat-dependent manner, a process that reflects the accessibility of the protein to the signal peptidase in the periplasm due to translocation across the cytoplasmic membrane (8, 10, 15, 34, 37, 68, 77, 80). To facilitate detection of the putative Tat substrates, the C termini of PetA and CcmE were tagged with the FLAG epitope. When PetA-FLAG was expressed in wild-type E. coli MC4100, a 24-kDa protein and an 18-kDa protein reacted with the anti-FLAG antibody, consistent with the predicted sizes of PetA before and after signal peptide cleavage, respectively (Fig. 1A). However, in the tatB-negative strain BØD, only the 24-kDa protein was detected (Fig. 1A), indicating that the processing of PetA-FLAG in E. coli depends on a functional Tat machinery. Similarly, in L. pneumophila, processing of PlcA-FLAG to its lower-molecular-weight form was detected in wild-type 130b but absent in the tatB mutant NU287 (Fig. 1B). When CcmE-FLAG was expressed in E. coli and L. pneumophila strains, only a single (17-kDa) protein was detected in both wild-type and tatB mutant strains (data not shown). Since the size of that protein was equivalent to the predicted size of CcmE with its signal peptide, CcmE-FLAG does not appear to be processed, at least under the conditions that we tested. Nonetheless, the results obtained with PetA-FLAG indicate that the Tat machinery is functional in L. pneumophila and that at least PetA is transported via this pathway. Additionally, the Tat dependency of the PetA cytochrome c reductase explains, at least in part, the Nadi-negative phenotype of the L. pneumophila tatB mutants.



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  		FIG. 1. Immunodetection of epitope-tagged PetA in total extracts of E. coli (A) and L. pneumophila (B). Bacterial extracts were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and reacted with the monoclonal anti-FLAG antibody. (A) Loading was as follows: lane 1, MC4100(pMMB2002); lane 2, BØD(pMMB2002); lane 3, MC4100(pMpetA); lane 4, BØD(pMpetA). (B) Loading was as follows: lane 1, 130b(pMMB2002); lane 2, 130b(pMpetA); lane 3, NU287(pMpetA). The highest-molecular-weight cross-reactive protein represents a nonspecific reaction, since it is present in the negative control in lane 1. Similar results were obtained in two additional and independent experiments.

Influence of tatB on L. pneumophila functions associated with type II secretion. Given that the Tat pathway of P. aeruginosa promotes the secretion of a subset of type II secreted proteins (46, 77), we sought to investigate whether the L. pneumophila tatB gene influences type II-dependent secretion in strain 130b. Thus, we first analyzed the culture supernatants of tatB-negative NU287 and NU288 for all the enzymatic activities that we have shown to be dependent on lspDE, lspF, lspG, and pilD (2, 3, 27, 39, 62, 63). The protease, tartrate-sensitive and tartrate-resistant acid phosphatase, RNase, PLA, LPLA, and lipase activities of the tatB mutants were comparable to those of the wild-type strain (Table 3). Thus, the tatB mutants do not have a general defect in the export of degradative enzymes. However, when we compared the culture supernatants of wild-type and tatB-negative strains for their ability to hydrolyze p-NPPC, a classical substrate for PLC enzymes of bacteria (4, 6, 46, 77), we observed that both tatB mutants NU287 and NU288 showed a 30% reduction in secreted PLC activity (Table 3). The introduction of tatB-expressing pMtatB into the mutants increased the p-NPPC hydrolase activity in the supernatants (Table 3). Taken together, these data indicate that L. pneumophila tatB influences the secretion of a PLC activity. In order to ascertain whether the tatB-associated PLC activity was also dependent on type II secretion, we used allelic exchange to introduce an lspF mutation into the NU288 tatB mutant. Two independent tatB lspF double mutants (i.e., NU289 and NU290) were obtained and tested for secreted enzymatic activities. When tested for p-NPPC hydrolase activity, supernatants of NU289 and NU290 were no more defective than those of the lspF mutant (Table 3 and data not shown), suggesting that the tatB mutation is altering the same pathway as the lspF mutation and that the tatB-dependent PLC uses the Lsp system for its ultimate secretion.


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  		TABLE 3. Secreted activities of L. pneumophila mutants

The close examination of the signal sequence of L. pneumophila PlcA, a secreted PLC of strain 130b that we previously described (4), revealed the presence of two arginines, i.e., MKKSIRRLVKPRLFAMSISLLAFS. This observation suggested that the secretion of PlcA might be dependent on the Tat pathway in L. pneumophila. When we compared the culture supernatants of tatB-negative strains and the plcA mutant, we observed that the reduction in secreted PLC activity in the tatB single mutant was not as great as that exhibited by a plcA mutant (Table 3). We therefore suspect that PlcA might be translocated by the Sec pathway in the absence of the Tat system; such a situation has been documented in Bacillus subtilis (35). To examine further the Tat dependency of PlcA, we attempted to monitor PlcA-FLAG processing, as was done for PetA-FLAG. When PlcA-FLAG expression was induced in E. coli MC4100, the expected ca. 49 kDa was detected with the anti-FLAG antibody (data not shown). However, the protein was not detected in L. pneumophila wild-type or the tatB mutant strains (data not shown), implying that PlcA-FLAG is unstable in L. pneumophila. We therefore cannot yet conclude that PlcA is a substrate of the Tat machinery in L. pneumophila.

Next, we examined the tatB-negative strains for altered colony color and morphology, another phenotype that we found to be associated with loss of type II secretion (39, 62, 63). Strains NU287 and NU288 produced colonies comparable to the wild type (data not shown), suggesting that the L. pneumophila tatB gene is not needed for Lsp-dependent modifications of the cell surface. We have recently observed that the L. pneumophila type II secretion system promotes bacterial growth at low temperatures, e.g., type II secretion mutants display a ca. 10,000-fold-reduced efficiency of plating on BCYE agar at 25°C (70). To test the possible involvement of tatB in this phenotype, wild-type 130b, the lspF mutant NU275, and the tatB mutants NU287 and NU288 were grown for 3 days on BCYE plates at 37°C, resuspended in BYE, and then replated for isolated colonies on BCYE agar and incubated at 37 and 25°C. As observed previously, after 8 days at 25°C, strain 130b produced 50% ± 8% of the CFU obtained at 37°C in 3 days and the lspF mutant yielded 0.002% ± 0.001% of the number CFU obtained at 37°C. The tatB mutants NU287 and NU288 exhibited an efficiency of plating at 25°C similar to that of the wild-type strain, i.e., 37% ± 14% and 36% ± 1%, respectively. Taken together, these data indicate that the L. pneumophila tatB gene influences a subset of the type II secretion-dependent phenotypes, i.e., the secretion of a PLC activity.

Influence of tatB on extracellular growth under iron-limiting conditions and siderophore production by L. pneumophila. Given our interest in L. pneumophila iron acquisition (40, 47, 59, 75, 76) and the fact that the Tat system promoted growth of P. aeruginosa in the presence of the iron chelator ethylene diamine diacetate (46), we next investigated the role of L. pneumophila tatB in extracellular growth under iron-limiting conditions. Towards that end, bacteria were grown for 3 days on standard BCYE agar, an iron-replete medium that is supplemented with 340 µM ferric pyrophosphate, and then resuspended in sterile water and spread onto BCYE agar with or without the added iron. As previously observed (59, 76), the parental strain 130b yielded a comparable number of colonies on the BCYE+Fe and BCYE-Fe plates (data not shown). Although the tatB-negative NU287 and NU288 also produced similar numbers of CFU on the two media after 3 days at 37°C, the colonies on BCYE-Fe agar were half the size of those on BCYE+Fe plates. Further depletion of the iron from the BCYE-Fe agar by the addition of 0.4 mM iron chelator DIP prevented colony formation by the tatB mutants after 3 days at 37°C, whereas the recovery of the wild type and an lspF-negative NU275 strain was not affected (Table 4). After another day of incubation, tatB mutant colonies appeared on the DIP-containing plates (Table 4). The growth defect of NU287 and NU288 under iron-limiting conditions that was manifest at 3 days was complemented by providing tatB in trans on pMtatB (Table 4). Consistent with the results obtained on solid media, the MIC of DIP in BYE broth was 0.125 mM for the tatB mutants versus 0.5 mM for parental strain 130b and lspF mutant NU275. The tatB mutants' growth defect in iron-limiting BYE was reversed by the addition of pMtatB. Thus, L. pneumophila tatB promotes extracellular growth under low-iron conditions.


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  		TABLE 4. Efficiency of plating of L. pneumophila strains on iron-depleted BCYE agar

In a recent study, we discovered that L. pneumophila produces a nonhydroxymate, nonphenolate siderophore (legiobactin) which is detectable with the CAS assay (40). To determine whether the reduced ability of the tatB mutants to grow under low-iron conditions was due to altered legiobactin production, NU287 was tested for the production of a CAS-reactive substance when grown in deferrated chemically defined medium. The tatB mutant produced levels of CAS reactivity that were comparable to those of wild type (data not shown), suggesting that iron acquisition processes other than legiobactin production are tatB dependent.

Intracellular infection of amoebae and macrophages by L. pneumophila tatB mutants. To determine the role of tatB in intracellular infection, we first compared the ability of strains 130b, lspF mutant NU275, and tatB mutants NU287 and NU288 to replicate within the protozoan host H. vermiformis under our standard conditions. In two different experiments, strains 130b, NU287, and NU288 behaved similarly, showing typical replication in which bacterial numbers increased ca. 1,000-fold in 96 h (Fig. 2). In contrast, and as previously described (63), the CFU recovered for the type II secretion mutant only increased ca. 10-fold over the course of the infection (Fig. 2). Thus, the tatB gene is not required for intracellular growth of L. pneumophila in H. vermiformis. To determine the role of tatB in replication within human cells, monolayers of U937 macrophages were infected with the wild-type 130b and the lspF and tatB mutants (Fig. 3A). In three independent experiments, the tatB mutants exhibited a ca. 10-fold reduction in CFU recovered from the monolayers 48 h postinfection (Fig. 3A). Since strains 130b, NU287, and NU288 survived equally well in the medium used to culture the macrophages (data not shown), this reduction in recovery was associated with a decrease in intracellular replication. The mutants' defect was fully complemented by providing tatB in trans on pMtatB (Fig. 3B), confirming a role for tatB in L. pneumophila infection of U937. In the same experiments, the lspF mutant exhibited an overall defect similar to that of the tatB-negative strains, although its reduced recoverability was first detectable at 24 h postinfection (Fig. 3A), as had been seen before (63).



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  		FIG. 2. Infection of amoebae with tatB mutants of L. pneumophila. Wells containing H. vermiformis were infected at a multiplicity of infection of 0.1 with wild-type 130b ({blacktriangleup}), lspF mutant NU275 ({square}), and tatB mutants NU287 ({circ}) and NU288 ({triangleup}). Bacterial CFU per well were determined at 0, 48, and 96 h after inoculation. Each datum point represents the mean and standard deviation (error bars) of three wells. Similar results were obtained in an additional experiment. At 48 and 96 h postinoculation, significant differences in recovery were obtained between 130b and NU275 (Student's t test; P < 0.001).



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  		FIG. 3. Infection of U937 cell macrophages with L. pneumophila tatB mutants and their complemented derivatives. Monolayers of U937 cells were infected at a multiplicity of infection of 0.1 for 2 h, washed to remove unincorporated bacteria, and then, 0, 24, and 48 h later, the monolayers were lysed and the total number of CFU in each well was determined. Results are the mean and standard deviation (error bars) of three wells and are representative of at least two independent experiments. (A) Wells containing U937 cells were infected with wild-type 130b ({diamondsuit}), lspF mutant NU275 ({square}), and tatB mutants NU287 ({circ}) and NU289 ({triangleup}). At 24 and 48 h postinoculation, significant differences in recovery were obtained between 130b and NU275 (Student's t test; P < 0.001). The differences in recovery between wild type and the tatB mutants were significant at 48 h (Student's t test; P < 0.001). (B) U937 monolayers were infected with strains 130b(pMMB2002) ({diamond}), 130b(pMtatB) ({diamondsuit}), NU287(pMMB2002) ({circ}), NU287(pMtatB) (•), NU288(pMMB2002) ({triangleup}), and NU288(pMtatB) ({blacktriangleup}). At 48 h postinoculation, significant differences in recovery were obtained between both NU287(pMMB2002) and NU288(pMMB2002) and the other four strains tested (Student's t test; P < 0.001).

Since tatB promoted the secretion of at least one type II-dependent enzymatic activity in L. pneumophila, we examined whether the intracellular growth defect of the tatB mutants in macrophages was due to the influence of tatB on type II secreted proteins. Towards that end, the tatB lspF mutant NU289 was tested in the intracellular infection assays. When examined in amoebae, the tatB lspF-negative strain was no more defective than was the lspF mutant NU275 (Fig. 4A), suggesting that the tatB mutation does not elicit a replication defect distinct from that generated by a type II secretion mutation. Upon infection of U937 cells, both strain NU289 and the lspF mutant NU275 exhibited a ca. 10-fold reduction of CFU compared to the wild type at 24 h (Fig. 4B). However, at 48 h postinoculation, there was a ca. 40-fold reduction in CFU recovery of NU289, whereas the reductions for the lspF-negative strain and the tatB-negative strain were only ca. 9-fold (Fig. 4B). That a mutant lacking both tatB and type II secretion is more defective than strains with single mutations was also evident when a tatB pilD mutant (i.e., strain NU299) was examined (Fig. 3B). These data suggest that the L. pneumophila tatB gene promotes intracellular infection of U937 cells by processes that are independent of type II secretion. The fact that the loss of PlcA, the only type II exoenzyme thus far linked to Tat, does not diminish L. pneumophila infectivity for U937 or amoebae (4) is supportive of this hypothesis.



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  		FIG. 4. Intracellular infection of amoebae and macrophages with L. pneumophila mutants defective for both tat and type II secretion genes. H. vermiformis (A) and U937 cells (B) were inoculated at a multiplicity of infection of 0.1, and then the numbers of bacteria in each well were determined at various times postinoculation. Results are the means and standard deviations (error bars) of triplicate wells and are representative of two independent experiments. (A) Infection profiles in amoebae for wild-type 130b ({diamondsuit}), lspF mutant NU275 ({square}), and tatB lspF mutant NU289 ({blacktriangleup}). At 46 and 70 h postinoculation, significant differences in recovery were obtained between wild type and both NU275 and NU289 (Student's t test; P < 0.001). (B) Infection profiles in U937 macrophages for wild-type strain 130b ({diamondsuit}), lspF mutant NU275 ({square}), tatB mutant NU288 ({triangleup}), tatB lspF mutant NU289 ({blacktriangleup}), and tatB pilD mutant NU299 (•). At 48 h postinoculation, significant differences in recovery were obtained between NU275 and NU289, NU275 and NU299, NU288 and NU289, and between NU288 and NU299 (Student's t test; P < 0.001).

Role of tatB in L. pneumophila intracellular infection under iron-limiting conditions. Since tatB promoted extracellular growth under iron-limited conditions, we tested the effect of iron depletion on the intracellular growth of tatB mutants in macrophages and protozoa. The role of tatB in intracellular growth in the presence of DIP was first tested in U937 cells. Upon treatment of U937 cells with 0, 25, and 50 µM DIP, a concentration range that does not adversely affect their viability (59), there was always a 15-fold reduction in CFU recovered for the tatB mutant NU288 compared to that with the wild type (Fig. 5). In the same experiment, the lspF mutant NU275 exhibited a sixfold reduction in recovery compared to 130b regardless of the concentration of DIP (Fig. 5). Thus, the reduced recovery of the tatB mutant as well as the lspF mutant in infected macrophages was not exacerbated by chelating iron with DIP. Conversely, adding additional iron to the macrophage cultures did not stimulate mutant growth (data not shown). Next, we performed cocultures of L. pneumophila and H. vermiformis in the presence of DIP, with concentrations of iron chelator that do not adversely affect viability of the amoebae (59). Wild-type 130b grew to similar levels in the presence of 0 and 25 µM DIP (Fig. 6A). In contrast, at 72 h postinoculation, the tatB mutant NU288 exhibited a ca. 20-fold reduction in CFU in cocultures containing 25 µM DIP (Fig. 6A). In the presence of 50 µM DIP, the intracellular growth of the wild type was diminished, with CFU numbers increasing 60-fold by 72 h (Fig. 6A). However, the tatB mutant NU288 did not have any significant increase in CFU in amoebae treated with 50 µM DIP (Fig. 6A). Since strains 130b and NU288 survived equally well in the medium used to culture the amoebae regardless of the concentration of DIP used (data not shown), the decreased recoverability of the tatB mutant is associated with impaired intracellular infection. Complementation of the growth defect of strain NU288 in DIP-treated cocultures was achieved with pMtatB (Fig. 6B), indicating that tatB promotes L. pneumophila intracellular growth in protozoa under iron-limiting conditions.



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  		FIG. 5. Replication of L. pneumophila tatB mutants within DIP-treated U937 macrophages. Macrophages were treated with 0, 25, or 50 µM DIP and then infected at a multiplicity of infection of 0.1 with wild-type 130b (black bars), tatB mutant NU288 (white bars), and lspF mutant NU275 (grey bars). Bacterial CFU per well were determined at 0 and 48 h after inoculation. Each datum point represents the mean and standard deviation (error bars) of three wells. Similar results were obtained in another experiment. Under all conditions tested, significant differences in recovery were obtained between 130b and NU288 and between 130b and NU275 (Student's t test; P < 0.001).



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  		FIG. 6. Growth of L. pneumophila tatB mutants within DIP-treated H. vermiformis. (A) Using a multiplicity of infection (MOI) of 0.1, wild-type 130b (diamonds) and tatB mutant NU288 (triangles) strains were used to infect amoebae that were treated with 0 (black symbols), 25 (gray symbols), or 50 µM DIP (white symbols). Bacterial CFU per well were determined at various time after inoculation. Data represent the mean and standard deviation from triplicate samples. At 71 h postinoculation, there was a significant difference in recovery between 130b and NU288 in cocultures treated with 25 and 50 µM DIP (Student's t test; P < 0.001). Similar results were obtained in an additional experiment, with the exception that NU288 did not show even the slightest increase in CFU in the presence of 50 µM DIP. (B) H. vermiformis cultures were treated with 0 or 25 µM DIP and then infected at an MOI of 0.1 with 130b(pMMB2002) (white bars), 130b(pMtatB) (black bars), NU288(pMMB2002) (stripped bars), and NU288(pMtatB) (gray bars). Bacterial CFU per well were determined at 0 and 72 h after inoculation. Each datum point represents the mean and standard deviation (error bars) of three wells. At 72 h postinoculation, significant differences in CFU recovery were observed between 130b(pMMB2002) and NU288(pMMB2002), as well as between NU288(pMMB2002) and NU288(pMtatB) (Student's t test; P < 0.01). In contrast, there were no significant differences in CFU recovery between the 130b cultures and NU288(pMtatB) (Student's t test; P > 0.05).


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DISCUSSION
 
In this study, we have identified the L. pneumophila locus encoding proteins homologous to E. coli TatA and TatB in strain 130b (67, 78). The presence of tatA and tatB as well as tatC is also evident in the recently reported complete genome of L. pneumophila strains Philadelphia-1, Paris, and Lens (11, 12, 16), suggesting that L. pneumophila encodes a twin-arginine pathway. Furthermore, reverse transcription-PCR analysis has confirmed the expression of tatA, tatB, and tatC in strain Philadelphia-1 (16). Our initial observation that the L. pneumophila tatB gene partially complemented the cell septation defect of the E. coli tatB mutant indicated that the Legionella tatB gene functions in twin-arginine translocation. This was confirmed by the analysis of the processing of PetA, a putative Tat substrate in L. pneumophila. Indeed, in L. pneumophila, as well as in recombinant E. coli, the cleavage of the PetA signal peptide was dependent on tatB.

L. pneumophila tatB mutants grew similar to wild-type bacteria in standard BYE broth and BCYE agar, indicating that the mutants' phenotype was not the result of a generalized growth defect. When grown aerobically, Agrobacterium tumefaciens, P. aeruginosa, and E. coli tat mutants exhibit replication, filamentation, and/or cell septation defects (18, 46, 71), a phenotype that, in E. coli, is associated with the loss of two cell wall-modifying amidases (8, 34). However, as in L. pneumophila, such defects were not observed for a Rhizobium leguminosarum tat mutant (43), suggesting that they are not always associated with the loss of the Tat system. When grown extracellularly under iron-depleted conditions, the L. pneumophila tatB mutants replicated more slowly than the wild type. Similarly, a P. aeruginosa Tat mutant failed to grow in the presence of an iron chelator, a phenotype which correlates with the observation that Tat function is essential for siderophore biosynthesis and uptake (46). Since our tatB mutants produced normal levels of siderophore, we suspect that the L. pneumophila tatB gene promotes iron acquisition independently from siderophore biosynthesis and export. Because legionellae are strict aerobes (24), we could only test the L. pneumophila tat mutants under aerobic conditions. Several studies have documented that tat mutants of gram-negative organisms are defective for anaerobic growth in the presence of specific respiratory substrates, consistent with the loss of periplasmic proteins with redox cofactors (7, 10, 18, 46, 67, 78). Our screen for possible Tat substrates identified PetA, a putative inner membrane-bound and periplasm-oriented respiratory subunit, i.e., a Rieske iron-sulfur subunit of the ubiquinol-cytochrome c reductase complex. Furthermore, we showed that the cleavage of the signal peptide of PetA was dependent on tatB both in E. coli and in L. pneumophila, establishing that PetA is a substrate of the Tat pathway. Homologs of this protein in A. tumefaciens and R. leguminosarum were also identified in a similar screen for possible Tat substrates (18, 43), a prediction that was experimentally confirmed in R. leguminosarum (43). Another candidate from our examination of the database was L. pneumophila CcmE, a putative periplasmic chaperone for cytochrome c maturation (76). Our attempt to confirm that CcmE was a Tat substrate was unsuccessful, since the signal peptide of the epitope-tagged protein did not appear to be processed in either E. coli or L. pneumophila strains. Nevertheless, our finding that PetA is a Tat substrate suggests that the L. pneumophila tatB gene influences cytochrome c-dependent respiration. Such a prediction was confirmed using the Nadi assay, a test for the activity of cytochrome c oxidase, which was negative for the L. pneumophila tatB mutants unless tatB was expressed in trans. Similarly, the R. leguminosarum tat mutant is negative in the Nadi assay, presumably because of the loss of ubiquinol-cytochrome c reductase activity (43).

Our mutational analysis has shown that the L. pneumophila tatB gene also facilitates secretion of PLC activity, a phenotype also associated with the Tat system in P. aeruginosa (46, 77). In this latter organism, two secreted PLCs, PlcH and PlcN, bear twin arginines in their signal peptides and the site-directed mutagenesis of one of the arginines abolishes secretion of PlcH (46). Moreover, analyses of bacterial genomes have uncovered that five homologs of PlcH and PlcN have a typical Tat signal sequence (17), suggesting that PLCs may be substrates of the Tat system in other organisms as well. Although our examination of the L. pneumophila genomes did not identify any homologs of this PLC family, L. pneumophila possesses a gene (i.e., plcA) that promotes secretion of PLC activity and whose product is homologous to another class of PLC identified in Pseudomonas fluorescens (4, 56). Most notably, the L. pneumophila PlcA proteins of strains 130b and Lens bear twin arginines in their signal peptides (4, 11), suggesting that secretion of PlcA depends on the Tat system. Although we did observe a reduction in PLC activity in the supernatants of tatB mutants, it was not as marked as the reduction observed for a plcA mutant, suggesting that a fraction of PlcA is secreted independently of tatB. We suspect that PlcA may be translocated by the Sec pathway in the absence of the Tat system, a situation that has been documented for the lipase LipA of B. subtilis (35). Indeed, the signal peptide of PlcA in strains 130b and Lens does not have a positively charged residue(s) near its C-terminal end, which acts as a Sec avoidance signal (9, 15). Moreover, examination of the genome sequences of L. pneumophila Philadelphia-1 and Paris (11, 12) reveals that the PlcA proteins in these strains do not bear a twin arginine in their N terminus and must therefore rely solely upon the Sec system for translocation. Our attempt to further examine the Tat dependency of PlcA was hindered by the apparent instability of the epitope-tagged PlcA-FLAG in L. pneumophila. As an alternate hypothesis to explain the reduced PLC activity in L. pneumophila tatB mutant supernatants, it is possible that tatB plays a role in the secretion of a yet-unidentified PLC. Nevertheless, supernatant analysis of the tatB lspF double mutants suggests that the L. pneumophila tatB gene promotes the secretion of a PLC activity that is also dependent on type II secretion. Other than PLC secretion, none of the known Lsp-dependent phenotypes in L. pneumophila observable in BYE broth or agar (i.e., secretion of other degradative enzymes, colony morphology, and growth at low temperature) were affected by the tatB mutation. In P. aeruginosa, whereas the secretion of PlcH and PlcN is tat dependent, two other type II exoproteins are secreted independently of the Tat pathway (77).

Our in vitro infection data demonstrate, for the first time, the importance of a tatB gene for optimal intracellular infection of human host cells. The L. pneumophila tatB mutants exhibited a 10-fold decrease in CFU recovery from infected human U937 cell macrophages. Importantly, the defect in macrophage infectivity could be complemented by providing tatB in trans. Although the role of the Tat system has been mainly studied in extracellular bacteria, one report established the importance of R. leguminosarum tat genes for the establishment of symbiosis with pea roots (43). To test whether the role of tatB in human macrophage infection was due to the link between tatB and type II secretion, L. pneumophila strains mutated in both tatB and type II secretion genes were tested in U937 cells. Double mutants were more defective than strains carrying a single mutation in either tatB or type II secretion genes, suggesting that tatB promotes intracellular infection of human cells by a mechanism that is independent of Lsp secretion. When tested for replication within amoebae under standard coculture conditions, which requires a greater participation from the type II secretion system than in macrophages, the L. pneumophila tatB mutants exhibited normal intracellular growth. These results are compatible with the finding that several mutants with reduced secreted PLC activity, but normal secretion of other exoenzymes, are not affected in intracellular infections of human cells and amoebae (3, 4). A defect in recovery of the tatB mutant was noted only in iron-depleted H. vermiformis amoebae. The differences in the recovery of the tatB mutants in macrophages and amoebae under iron-replete conditions suggest that this gene is more important in macrophages than in amoebae, or that intracellular iron availability may be more limiting in macrophages than in H. vermiformis. Such observations were also made for the L. pneumophila feoB mutant, which is defective in ferrous iron uptake (59). Therefore, the L. pneumophila tatB gene most probably influences factors, e.g., growth under low-iron conditions, that are important for human cell infection and to a lesser extent for amoebae infection. In particular, our lab has previously shown that mutations in L. pneumophila ccmC result in changes in phenotype that resemble those of the tatB mutants, i.e., loss of cytochrome c-dependent respiration, impaired ability to grow extracellularly under low-iron conditions, and a reduction in the ability to grow within macrophages and amoebae (76). However, the last two phenotypes of the ccmC mutants are more severe than those observed for the tatB mutants, which could reflect the central role of a functional cytochrome c, rather than its redox state, in these processes. Alternatively, it is possible that the ubiquinol-cytochrome c reductase plays a more important role in intracellular rather than extracellular growth. Finally, other potential L. pneumophila Tat substrates (Table 2) may facilitate intracellular infections. Based on their homologies, the Tat substrates could be involved in processes as diverse as membrane bioenergetics, substrate binding and transport, metabolism of lipids, amino acids, and nucleic acids, cell division, and adaptation to atypical conditions (Table 2). In summary, we have demonstrated that the L. pneumophila tatB gene promotes translocation of a putative redox protein across the inner membrane, secretion of PLC activity, growth under iron-limiting conditions, and intracellular infection. The Tat substrates that carry out these various processes will be the subject of future investigations.


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ACKNOWLEDGMENTS
 
We thank Kimberly Allard for her expert assistance with the CAS assay and Ben Billips for his help with some of the enzymatic assays. We acknowledge George Georgiou for providing E. coli strain BØD and Mecky Pohlschröder for providing the TATFIND 1.2 algorithm, as well as Sheng Yang He and Sruti DebRoy for the gift of plasmid pFLAG-CTC. We also thank past and present members of the Cianciotto lab for helpful discussions.

This work was supported by NIH grant AI43987 awarded to N.P.C. and by the Center for Genetic Medicine at Northwestern University for a travel award to O.R.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Northwestern University Medical School, 320 East Superior St., Chicago, IL 60611. Phone: (312) 503-0385. Fax: (312) 503-1339. E-mail: n-cianciotto{at}northwestern.edu. Back

Editor: D. L. Burns


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