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The mechanisms by which Legionella pneumophila, a facultative intracellular parasite and the agent of Legionnaires' disease, acquires iron are largely unexplained. Several earlier studies indicated that L. pneumophila does not elaborate siderophores. However, we now present evidence that supernatants from L. pneumophila cultures can contain a nonproteinaceous, high-affinity iron chelator. More specifically, when aerobically grown in a low-iron, chemically defined medium (CDM), L. pneumophila secretes a substance that is reactive in the chrome azurol S (CAS) assay. Importantly, the siderophore-like activity was only observed when the CDM cultures were inoculated to relatively high density with bacteria that had been grown overnight to log or early stationary phase in CDM or buffered yeast extract. Inocula derived from late-stationary-phase cultures, despite ultimately growing, consistently failed to result in the elaboration of siderophore-like activity. The Legionella CAS reactivity was detected in the culture supernatants of the serogroup 1 strains 130b and Philadelphia-1, as well as those from representatives of other serogroups and other Legionella species. The CAS-reactive substance was resistant to boiling and protease treatment and was associated with the <1-kDa supernatant fraction. As would also be expected for a siderophore, the addition of 0.5 or 2.0 microM iron to the cultures repressed the expression of the CAS-reactive substance. Interestingly, the supernatants were negative in the Arnow, Csáky, and Rioux assays, indicating that the Legionella siderophore was not a classic catecholate or hydroxamate and, hence, might have a novel structure. We have designated the L. pneumophila siderophore legiobactin.

In this study, we assessed the activity of ciprofloxacin, levofloxacin, sparfloxacin, and trovafloxacin against clinical isolates of Streptococcus pneumoniae that were resistant to the less-recently developed fluoroquinolones by using defined amino acid substitutions in DNA gyrase and topoisomerase IV. The molecular basis for resistance was assessed by using mutants selected with trovafloxacin, ciprofloxacin, and levofloxacin in vitro. This demonstrated that the primary target of trovafloxacin in S. pneumoniae is the ParC subunit of DNA topoisomerase IV, similar to most other fluoroquinolones. However, first-step mutants bearing the Ser79-->Phe/Tyr substitution in topoisomerase IV subunit ParC were susceptible to trovafloxacin with a minimum inhibitory concentration of 0.25 microg/ml, and mutations in the structural genes for both topoisomerase IV subunit ParC (parC) and the DNA gyrase subunit (gyrA) were required to achieve levels of resistance above the breakpoint. The data also suggest that enhanced activity of trovafloxacin against pneumococci is due to a combination of factors that may include reduced efflux of this agent and an enhanced activity against both DNA gyrase and topoisomerase IV.

Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative bacteria. To begin to assess the importance of type II secretion for the virulence of an intracellular pathogen, we examined the effect of inactivating the prepilin peptidase (pilD) gene of Legionella pneumophila. Although the pilD mutant and its parent grew similarly in bacteriological media, they did differ in colony attributes and recoverability from late stationary phase. Moreover, at least three proteins were absent from the mutant's supernatant, indicating that PilD is necessary for the secretion of Legionella proteins. The absence of both the major secreted protein and a haemolytic activity from the mutant signalled that the L. pneumophila zinc metalloprotease is excreted via type II secretion. Most interestingly, the pilD mutant was greatly impaired in its ability to grow within Hartmannella vermiformis amoebae and the human macrophage-like U937 cells. As reintroduction of pilD into the mutant restored inefectivity and as a mutant lacking type IV pilin replicated like wild type, these data suggested that the intracellular growth of L. pneumophila is promoted by proteins secreted via a type II pathway. Intratracheal inoculation of guinea pigs revealed that the LD50 for the pilD mutant is at least 100-fold greater than that for its parent, and the culturing of bacteria from infected animals showed a rapid clearance of the mutant from the lungs. This is the first study to indicate a role for PilD and type II secretion in intracellular parasitism.

The bacterium Legionella pneumophila is the responsible agent for Legionnaires' disease and has recently been shown to harbor a gene encoding a kinase that confers resistance to the aminoglycoside antibiotic spectinomycin (Suter, T. M., Viswanathan, V. K., and Cianciotto, N. P. (1997) Antimicrob. Agents Chemother. 41, 1385-1388). We report the overproduction, purification, and characterization of this spectinomycin kinase from an expressing system in Escherichia coli. The purified protein shows stringent substrate specificity for spectinomycin with Km = 21.5 microM and kcat = 24.2 s-1 and does not bind other aminoglycosides including kanamycin, amikacin, neomycin, butirosin, streptomycin, or apramycin. Purification of spectinomycin phosphate followed by characterization by mass spectrometry and 1H, 13C, and 31P NMR established the site of phosphorylation to be at the hydroxyl group at position 9. Thus this enzyme is designated APH(9)-Ia (where APH is aminoglycoside kinase). The enzyme was inactivated by the electrophilic ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine, consistent with a nucleophilic residue such as Lys lining the nucleotide binding pocket. Site-directed mutagenesis of Lys-52 and Asp-212 to Ala confirmed that these residues were important for catalysis, with Lys-52 playing a potential role in ATP binding and Asp-212 in phosphoryl transfer. Thio and solvent isotope effect experiments in the presence of either Mg2+ or Mn2+ were consistent with a kinetic mechanism in which phosphate transfer does not contribute significantly to the rate-limiting step. These results establish that APH(9)-Ia is a highly specific antibiotic resistance kinase and provides the requisite mechanistic information for future structural studies.

Previously, we had isolated by transposon mutagenesis a Legionella pneumophila mutant that appeared defective for intracellular iron acquisition. While sequencing in the proximity of the mini-Tn10 insertion, we found a locus that had a predicted protein product with strong similarity to PilB from Pseudomonas aeruginosa. PilB is a component of the type II secretory pathway, which is required for the assembly of type IV pili. Consequently, the locus was cloned and sequenced. Within this 4-kb region were three genes that appeared to be organized in an operon and encoded homologs of P. aeruginosa PilB, PilC, and PilD, proteins essential for pilus production and type II protein secretion. Northern blot analysis identified a transcript large enough to include all three genes and showed a substantial increase in expression of this operon when L. pneumophila was grown at 30 degrees C as opposed to 37 degrees C. The latter observation was then correlated with an increase in piliation when bacteria were grown at the lower temperature. Southern hybridization analysis indicated that the pilB locus was conserved within L. pneumophila serogroups and other Legionella species. These data represent the first isolation of type II secretory genes from an intracellular parasite and indicate that the legionellae express temperature-regulated type IV pili.

Non-toxigenic strains of Corynebacterium diphtheriae continue to cause disease within immunized populations. A subset of these corynebacteria carry the diphtheria toxin gene but in a cryptic form. To determine whether such strains might contribute to the re-emergence of functional toxin genes, the phages and tox mutations within three clone types were examined. tox-containing, beta-related phages were isolated from two of the strain types. The third isolate appeared to harbour a defective prophage. One of the tox- phages encoded truncated, yet enzymatically-active, forms of diphtheria toxin, suggesting that it had sustained a point mutation within the latter half of its toxin gene. In contrast, the other mutant phage did not elicit the production of either a cross-reacting material or an ADP-ribosylating activity. Complementation tests employing a series of double lysogens confirmed that the mutations responsible for the non-toxigenic phenotype of all of the phages were cis dominant. Given these findings, it is reasonable to hypothesize that tox+ genes can arise within human populations by either homologous recombination between two distinct tox- phages or spontaneous reversion within a single mutant allele.

A gene capable of conferring spectinomycin resistance was isolated from Legionella pneumophila, the agent of Legionnaires' disease. The gene (aph) encoded a 36-kDa protein which has similarity to aminoglycoside phosphotransferases. Biochemical analysis confirmed that aph encodes a phosphotransferase which modifies spectinomycin but not hygromycin, kanamycin, or streptomycin. The strain that was the source of aph demonstrated resistance to spectinomycin, and Southern hybridizations determined that aph also exists in other legionellae.

Legionella pneumophila, a parasite of alveolar macrophages, requires iron for intra- and extracellular growth. Although its mechanisms for iron assimilation are poorly understood, this bacterium produces Fur, a protein that can repress gene transcription in response to iron concentration. Because iron- and Fur-regulated genes are important for infection in other bacteria, the identification of similar genes in L. pneumophila was undertaken. A wild-type strain of L. pneumophila was randomly mutated with a mini-Tn10' lacZ transposon, and the resulting gene fusions were tested for iron regulation by assessing beta-galactosidase production in the presence and absence of iron chelators. Of the initial six mutants with iron-repressed lacZ fusions, two strains, NU229 and NU232, possessed fusions that were stably iron regulated. To assay for Fur regulation, the levels of beta-galactosidase were measured in strains no longer producing Fur. As in a number of pathogenic bacteria, L. pneumophila fur could not be insertionally inactivated, but spontaneous Fur- derivatives were generated by selecting for manganese resistance. Strain NU229 contained a Fur-repressed fusion based on derepression of lacZ expression in its manganese-resistant derivative. Extracellular growth of NU229 in bacteriological media was similar to that of wild-type strain 130b. To assess the role of an iron- and Fur-regulated (frgA) gene in intracellular infection, the ability of NU229 to grow within U937 cell monolayers was tested. Quantitative infection assays demonstrated that intracellular growth of NU229 was impaired as much as 80-fold. Reconstruction of the mutant by allelic exchange proved that the infectivity defect in NU229 was due to the inactivation of frgA and not to a second-site mutation. Subsequently, complementation of the interrupted gene by an intact plasmid-encoded gene demonstrated that the infectivity defect was due to the loss of frgA and not to a polar effect. Nucleotide sequence analysis revealed that the 63-kDa FrgA protein has homology with the aerobactin synthetases IucA and IucC of Escherichia coli, raising the possibility that L. pneumophila encodes a siderophore which is required for optimal intracellular replication. Southern hybridization analysis determined that frgA is specific to L. pneumophila.

We assessed the ability of Legionella species that have not been associated with disease to infect macrophage-like U937 cells. Two of fourteen species tested exhibited a 50% infective dose that was within I log unit of that of virulent L. pneumophila. Indeed, intracellular CFU of L. jamestowniensis and L. parisiensis increased 100-fold over a 72-h period. These data indicate that additional legionellae can flourish within phagocytes and therefore, can, if given the opportunity, cause disease.

Unlike most beta-related phages isolated from Corynebacterium diphtheriae, phage 782 readily plaqued on strains of C. ulcerans and C. pseudotuberculosis. The extended host range of phage 782 was not, however, due to a greater ability of the phage to absorb to bacterial surfaces. Using chemical mutagenesis, a number of phage mutants were isolated which had diminished capacities to infect C. ulcerans, suggesting the existence of a locus (ehr) for extended host range. Pre-lysogenization of C. ulcerans strains with phage 782, but not its mutant form or other beta-related phages, rendered them susceptible to infection by previously excluded phages. An examination of recombinant between phages 782, pie, and beta localized ehr to a 7 kilobase region of DNA including attP. The data are compatible with the notion that ehr encodes an anti-restriction function.

Conflicting reports have been given as to the existence of a Legionella pneumophila siderophore. Hence, we rigorously examined the reported siderophore-like activity using the chrome azurol S indicator. Although chrome azurol S reactivity was detected in supernatants, control experiments indicate that it was due to cysteine in the media. When bacteria were cultured in media without cysteine, no siderophores were detected.

The ability to bind and utilize hemin is a trait common to many human pathogens. Nevertheless, the relationship between Legionella pneumophila, the agent of Legionnaires' disease, and hemin has received little attention. Thus, we explored the capacity of a virulent, serogroup 1 strain of L. pneumophila to bind hemin and use it as an iron source. Hemin, but not protoporphyrin IX, restored bacterial growth in iron-limiting media, indicating that it can serve as an iron source for L. pneumophila. In support of this idea, we observed that wildtype legionellae were able to bind 50 to 60% of added hemin, a binding capacity that was comparable to those of other pathogens. To begin to identify proteins involved in hemin acquisition, we identified a Legionella locus that conferred hemin binding upon Escherichia coli. Subcloning and nucleotide sequence analysis determined that a single open reading frame, which was designated hbp for hemin-binding promotion, was responsible for this binding activity. The hbp gene was predicted to encode a secreted, 15.5-kDa protein. To ascertain the importance of this gene in L. pneumophila biology, we used allelic exchange to construct an hbp mutant. Importantly, the mutant displayed a 42% reduction in hemin binding, confirming that hbp potentiates hemin acquisition by L. pneumophila. However, the strain was unaltered in its ability to grow within macrophage-like cells and freshwater amoebae, indicating that hbp is not required for intracellular infection. Despite this, Southern hybridization analysis and database searches demonstrated that hbp is nearly exclusive to the L. pneumophila species.

Legionella pneumophila, a parasite of macrophages and protozoa, requires iron for optimal extracellular and intracellular growth. However, its mechanisms of iron acquisition remain uncharacterized. Using mini-Tn10 mutagenesis, we isolated 17 unique L. pneumophila strains which appeared to be defective for iron acquisition and assimilation. Eleven of these mutants were both sensitive to the iron chelator ethylenediamine di(o-hydroxyphenylacetic acid) and resistant to streptonigrin, an antibiotic whose lethal effect requires high levels of intracellular iron. Six mutants were also defective for the infection of macrophage-like U937 cells. Although none were altered in entry, mutants generally exhibited prolonged lag phases and in some cases replicated at slower rates. Overall, the reduced recoveries of mutants, relative to that of the wild type, ranged from 3- to 1,000-fold. Strain NU216, the mutant displaying the most severe lag phase and the slowest rate of replication, was studied further. Importantly, within U937 cells, NU216 was approximately 100-fold more sensitive than the wild type was to treatment with the Fe3+ chelator deferoxamine, indicating that it is defective for intracellular iron acquisition and assimilation. Furthermore, this strain was unable to mediate any cytopathic effect and was impaired for infectivity of an amoebal host. Taken together, the isolation of these mutants offers genetic proof that iron acquisition and assimilation are critical for intracellular infection by L. pneumophila.

The gene encoding a protein that reacted with antibodies specific for Legionella pneumophila macrophage infectivity potentiator (LpMip) was cloned from Coxiella burnetii, the obligate intracellular rickettsia that causes Q fever in humans. Nucleotide sequencing analysis revealed an ORF encoding a gene product of 230 amino acids with a molecular mass of 25.5 kDa and a predicted pI of 10.7. The predicted amino acid sequence from the ORF shows similarity with Mip/Mip-like proteins of Legionella (46%) and Chlamydia (30%). Moreover, like LpMip, the amino acid sequence of the C terminus of this protein has over 35% identity to prokaryotic and eukaryotic FK506-binding proteins (FKBPs) that belong to a superfamily of immunophilins and are peptidyl-prolyl cis-trans isomerases (PPIases). When overproduced in Escherichia coli, the C. burnetii protein also exhibited PPIase activity. Taken together, these results demonstrate that C. burnetii encodes a Mip analogue (CbMip). A putative leader peptide at the N terminus of CbMip was detected by computer analysis. Furthermore, TnphoA mutagenesis demonstrated that in E. coli CbMip was secreted. In view of the role of Mip/Mip-like proteins in the pathogenesis of Legionella and Chlamydia, CbMip may be a C. burnetii virulence factor.

The pathogenesis of Legionella micdadei is dependent upon its ability to infect alveolar phagocytes. To better understand the basis of intracellular infection by this organism, we examined the importance of its Mip surface protein. In Legionella pneumophila, Mip promotes infection of both human macrophages and freshwater protozoa. Southern hybridization and immunoblot analyses demonstrated that mip sequences were present and expressed within a panel of virulent L. micdadei strains. Using allelic exchange mutagenesis, we then constructed an L. micdadei strain that completely and specifically lacked Mip. Although unimpaired in its ability to grow in bacteriologic media, this Mip mutant was defective in its capacity to infect U937 cells, a human macrophage-like cell line. Most significantly, the Mip- organism displayed a 24-fold reduction in survivability immediately after its entry into the phagocyte. Similarly, the mutant was less able to parasitize Hartmannella amoebae. Taken together, these data argue that Mip specifically potentiates intracellular growth by L. micdadei.

Legionella pneumophila can invade and grow within explanted alveolar epithelial cells. Given its potential clinical significance, an examination of the molecular basis of epithelial cell infection was initiated. The mip gene encodes a 24-kilodalton surface protein that promotes macrophage infection and virulence. To determine whether this gene is required for pneumocyte infection, we tested a strain bearing a mip null mutation for its ability to infect both explanted type II cells and type I-like cell lines. For infection of type II cells, the infective dose 50% for the Mip-strain was 25-fold higher than an isogenic Mip+ strain. Type I cell monolayers infected with the mutant for 3 days yielded approximately 50-fold fewer bacteria than did monolayers infected with the parental strain. These data indicate that Mip enhances infection of pneumocytes and that L. pneumophila employs some of the same genes (mechanisms) to infect epithelial cells and macrophages.

The Mip surface protein, a prokaryotic analog of the FK506-binding proteins, enhances the ability of Legionella pneumophila to infect macrophages and protozoa. Using mip-specific probes and low-stringency Southern hybridizations, we have detected DNA sequences homologous to mip within Coxiella burnetii and Rochalimaea quintana. Using specific anti-Mip antisera and immunoblot analysis, we also detected Mip-related proteins within these bacteria as well as within Rickettsia and Ehrlichia species. These data suggest that Mip-related proteins have broad significance for host-parasite interactions. However, they also indicate that care must be exercised when using mip probes or anti-Mip antibodies for the detection of Legionella organisms in water or clinical samples.

To more effectively study the genetic basis of Legionnaires' disease, we characterized a system for mini-Tn10 mutagenesis in Legionella pneumophila. The mini-transposons were first electroporated into Legionella on counterselectable vectors expressing altered target site transposases. Then, by simultaneously selecting for the kanamycin-resistance gene within the transposon and counterselecting against the maintenance of the plasmid, we directly and readily isolated strains bearing single chromosomal insertions. Southern hybridization analysis further demonstrated that the insertions were randomly distributed throughout the Legionella genome. The mini-Tn10 insertions were stable during extracellular and intracellular growth, and did not alter the infectivity of L. pneumophila. Thus, this mutagenesis system offers an easy, one-step approach toward isolating large populations of random mutants which can be screened for defects in virulence.

Iron is required for the intracellular and extracellular growth of Legionella pneumophila (Lp). In addition, variations in iron levels may serve as a signal for changes in gene expression. In a number of bacterial pathogens, the regulation of gene expression by iron is usually mediated by the Fur (ferric uptake regulation) repressor protein. Through complementation of an Escherichia coli fur mutation and nucleotide sequence analysis, we have cloned and characterized the Lp fur gene. Lp fur encoded a 15.0-kDa protein whose repressive activity was, as expected, highest in bacteria grown in iron-rich media. Computer analysis determined that Lp Fur had an amino-acid identity of over 54% and a similarity of over 72% to the Fur of E. coli, Yersinia pestis, Vibrio species and Pseudomonas aeruginosa. The promoter region of Lp fur contained sequences homologous to the Fur-binding site, suggesting that fur is autoregulated in Lp. Finally, Southern blot hybridizations demonstrated that fur is conserved among Lp strains and Legionella species.

Legionella pneumophila is an intracellular parasite of freshwater protozoa and human macrophages. Recent studies determined that the macrophage infectivity potentiator (Mip) surface protein, a prokaryotic homolog of the FK506-binding proteins, is required for optimal infection of macrophages. To determine whether Mip is also involved in L. pneumophila infection of protozoa, we examined the ability of a strain lacking Mip to parasitize Hartmannella amoebae and Tetrahymena ciliates. After 3 days of incubation, approximately 1000-fold fewer bacteria were recovered from protozoan cocultures infected with the Mip- strain than from those cocultures infected with an isogenic Mip+ strain. The mip mutant was, however, not impaired in its ability to bind to amoebae cell surfaces, indicating that Mip is involved in bacterial resistance to intracellular killing and/or intracellular multiplication. These data suggest that L. pneumophila employs similar genes and mechanisms to infect human cells and protozoa. Furthermore, they support the hypothesis that the ability of L. pneumophila to parasitize macrophages and hence to cause human disease is a consequence of its prior adaptation to intracellular growth within protozoa.

After the demonstration of analogs of the Legionella pneumophila macrophage infectivity potentiator (Mip) protein in other Legionella species, the Legionella micdadei mip gene was cloned and expressed in Escherichia coli. DNA sequence analysis of the L. micdadei mip gene contained in the plasmid pBA6004 revealed a high degree of homology (71%) to the L. pneumophila mip gene, with the predicted secondary structures of the two Mip proteins following the same pattern. Southern hybridization experiments, with the plasmid pBA6004 as the probe, suggested that the mip gene of L. micdadei has extensive homology with the mip-like genes of several Legionella species. Furthermore, amino acid sequence comparisons revealed significant homology to two eukaryotic proteins with isomerase activity (FK506-binding proteins).

The mip gene of Legionella pneumophila serogroup 1 strain AA100 encodes a 24-kilodalton surface protein (Mip) and enhances the abilities of L. pneumophila to parasitize human macrophages and to cause pneumonia in experimental animals. To determine whether this virulence factor is conserved in the genus Legionella, a large panel of Legionella strains was examined by Southern hybridization and immunoblot analyses for the presence and expression of mip-related sequences. Strains representing all 14 serogroups of L. pneumophila contained a mip gene and expressed a 24-kilodalton Mip protein. Although the isolates of the 29 other Legionella species did not hybridize with mip DNA probes under high-stringency conditions, they did so at reduced stringency. In support of the notion that these strains possess mip-like genes, these species each expressed a protein (24 to 31 kilodaltons in size) that reacted with specific Mip antisera. Moreover, the cloned mip analog from Legionella micdadei encoded the cross-reactive protein. Thus, mip is conserved and specific to L. pneumophila, but mip-like genes are present throughout the genus, perhaps potentiating the intracellular infectivity of all Legionella species.

Infection by Legionella pneumophila is believed to depend upon its ability to multiply within host alveolar macrophages. To investigate this, a site-specific mutation was introduced into a gene (mip) that encodes a 24,000-Da surface protein; an 80-fold loss of infectivity for both U937 cells and explanted human alveolar macrophages was observed. Further phenotypic analysis of the mutant strain has failed to show alterations in bacterial factors (e.g., proteinase, lipopolysaccharide) that have suspected roles in virulence. To substantiate that this mutation also results in reduced virulence in animals, the lethality and clinical illnesses produced by the parent and mutant L. pneumophila strains were compared in guinea pigs after intratracheal inoculation. The mutant strain produced fewer illnesses, slower-progressing disease, and fewer lethal infections than either the parent strain or a derivative of the mutant strain with the wild-type mip gene reintroduced. When sublethal inocula of the three strains were used, the mutant bacteria were recovered in slightly lower numbers from lung homogenates and in significantly lower numbers from the spleen, at 48 h, than were the other two test strains. Thus mip seems to be necessary for full virulence of L. pneumophila and may represent the first genetically defined virulence factor in this species.

In a previous study, a 24-kilodalton (kDa) protein surface antigen of Legionella pneumophila was cloned into Escherichia coli and found to be expressed on the host cell surface. Subsequently, a site-directed mutation in this gene (designated mip) in L. pneumophila was found to impair the capacity of this bacterium to initiate intracellular infection in human macrophages. The work presented here indicates that the antigenic gene product is distinct from the 24- to 29-kDa major outer membrane protein of L. pneumophila. In addition, the antigen was identified as a highly basic protein on two-dimensional nonequilibrium polyacrylamide gels and on two-dimensional monoclonal antibody immunoblots. When the DNA fragment encoding this protein was sequenced, a long open reading frame of 699 base pairs was identified within a region to which antigen expression was previously mapped. mip mRNA isolated from both L. pneumophila and transformed E. coli had the same 5' end, as determined by primer extension analysis, indicating that the same promoter sequences are used in both species. A likely factor-independent transcriptional terminator was found 20 residues downstream of the stop codon, suggesting that mip is encoded on a monocistronic message. The inferred polypeptide began with a possible 20- to 24-residue signal sequence, and, as predicted by two-dimensional electrophoresis, had a molecular weight of 24,868 and was a potent polycation with an estimated pI of 9.8.

To investigate the pathogenesis of Legionnaires disease at a molecular level, we mutated by directed allelic exchange a gene encoding a Legionella pneumophila-specific 24,000-dalton (Da) surface protein. Southern hybridization and immunoblot analyses demonstrated that the predicted DNA rearrangement occurred in L. pneumophila with a specific loss of 24-kDa antigen expression. Compared with its isogenic parent, the mutant was significantly impaired in its ability to infect transformed U937 cells, a human macrophagelike cell line; i.e., the bacterial inoculum of the mutant strain that was required to initiate infection of the macrophage monolayer was ca. 80-fold greater than that of the isogenic parent strain. The mutant strain regained full infectivity on reintroduction of a cloned 24-kDa protein gene, indicating that the reduced infectivity was due specifically to the mutation in that gene. Compared with the parent strain, the mutant strain was recovered at titers that were ca. 10-fold lower shortly after infection, but it exhibited a similar intracellular growth rate over the next 40 h, indicating that the mutant was defective in its ability to initiate macrophage infection rather than in its ability to replicate intracellularly. When opsonized, the mutant strain was still significantly less infectious than the parent strain, despite equivalent macrophage association, suggesting that the mutant was not merely missing a ligand for macrophage attachment. The mutant also exhibited reduced infectivity in explanted human alveolar macrophages, demonstrating the relevance of the U937 cell model for analyzing this mutant phenotype. These results represent the first identification of a cloned L. pneumophila gene that is necessary for optimal intracellular infection; we designate this gene mip, for macrophage infectivity potentiator.