lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin

Abstract

Under iron stress, Legionella pneumophila secretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes, lbtA and lbtB, which are involved in legiobactin production. lbtA appears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein of L. pneumophila. lbtB encodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA or lbtB were defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2',2'-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. The lbtA mutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of the lbt mutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. The lbtA mutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However, L. pneumophila expresses lbtA in the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery of lbtAB represents the first identification of genes required for L. pneumophila siderophore expression.

FIG. 1.

Kinetics of siderophore production by L. pneumophila. Strain 130b bacteria were grown in BYE to an OD₆₆₀ of 1.0, washed, and then inoculated into deferrated CDM to an OD₆₆₀ of 0.2. Over the next day, the growth of the cultures was monitored spectrophotometrically (left y axis), and the CAS reactivity of culture supernatants, reported as net DFX equivalents, was examined (right y axis). The values presented are the means and standard deviations from triplicate cultures. The CAS reactivity of the cultures was significantly above the medium control at all times of incubation (P < 0.05; Student's t test). The results are characteristic of three independent experiments.

FIG. 2.

Effect of temperature on L. pneumophila siderophore expression. Strain 130b bacteria were grown at 37°C in BYE to an OD₆₆₀ of 1.0, washed, and then inoculated into deferrated CDM to an OD₆₆₀ of 0.2. Over the next 3 days, one set of cultures was incubated at room temperature (□), and another set at 37°C (⧫). At various time points, the growth of the cultures was monitored spectrophotometrically (top), and the CAS reactivity of culture supernatants was examined (bottom). The values presented represent the means and standard deviations from duplicate cultures. The CAS reactivity of the room temperature cultures was significantly different from that of the 37°C cultures at all times of incubation, except at 40 h (P < 0.05; Student's t test). The results presented are representative of four independent experiments.

FIG. 3.

Biological activities associated with legiobactin. (A) 10⁴ CFU of wild-type strain 130b were inoculated into non-iron-supplemented BCYE agar (leftmost plate) or non-iron-supplemented BCYE agar containing 600 μM DIP (four rightmost plates), and a center well was filled, as indicated, with 50 μl of either deferrated CDM, 10 mM FeCl₃, or the <3-kDa fraction from a CAS-negative (i.e., 194 μM DFX equivalents) or CAS-positive (i.e., 1,100 μM DFX equivalents) supernatant of strain 130b. After 21 days of incubation at room temperature, the growth of the bacteria was recorded. (B) We plated 10⁵ CFU of feoB mutant bacteria onto the surface of standard (i.e., iron-supplemented) BCYE agar (leftmost plate) or non-iron-supplemented BCYE agar (four rightmost plates), and a center well was filled, as indicated above, with either deferrated CDM, FeCl₃, or a CAS-negative or CAS-positive supernatant fraction. After 5 days of incubation at 37°C, the growth of the bacteria was recorded. The results presented for each type of bioassay are representative of at least three independent experiments.

FIG. 4.

Extra- and intracellular expression of L. pneumophila lbtA. (A) Wild-type 130b was grown on non-iron-supplemented BCYE agar for 3 days, subcultured into BYE lacking the iron supplement, and grown to an OD₆₆₀ of 1.0. Bacteria were then inoculated into deferrated CDM (CDM-Fe) or CDM supplemented with 20 μM FeCl₃ (CDM+Fe) at an OD₆₆₀ of 0.3 and incubated at 37°C. Bacteria were harvested at mid-log phase and the RNA was isolated. RT-PCR was performed to detect mip (M) and lbtA (L) transcripts. Lanes N, negative controls for DNA contamination performed without reverse transcriptase. (B) Bacteria were grown as in A in BYE-Fe to an OD₆₆₀ of 1.0. U937 cells were infected with log-phase bacteria and RNA was isolated from infected cells at 24, 48, and 72 h postinoculation. Between 0 and 24 h postinoculation, the numbers of legionellae increased 1,000-fold, and then between 24 and 72 h postinoculation, the numbers increased another 10-fold. Lane denotation is the same as in A. lbtA was also expressed intracellularly when a stationary-phase inoculum was used (data not shown). The results presented are representative of at least two independent experiments. RT-PCR products were electrophoresed through 1.5% agarose and stained with ethidium bromide.

FIG. 5.

Siderophore production by L. pneumophila wild type and lbtA and lbtB mutants. (A) (Left panel) Wild-type130b with pMMB2002 (⋄) or plbtA (⧫) and lbtA deletion mutant NU302 with pMMB2002 (▪) or plbtA (□) were grown in BYE to an OD₆₆₀ of 1.0, inoculated into deferrated CDM to an OD₆₆₀ of 0.3, and then incubated at 37°C. At various times, the growth of the cultures was monitored spectrophotometrically (data not shown), and the CAS reactivity of supernatants was examined. The CAS values presented represent the means and standard deviations from duplicate cultures. The CAS reactivity of the mutant was significantly different from that of the complemented mutant at all time points and wild-type cultures up to 22 h postinoculation (P < 0.05; Student's t test). The results presented are representative of at least three independent experiments. (Right panel) We plated 10⁵ CFU of feoB mutant bacteria onto the surface of non-iron-supplemented BCYE agar and a center well was filled with either a <3-kDa supernatant fraction from the lbtA mutant NU302 or a <3-kDa supernatant fraction from the complemented mutant NU302(plbtA). After 5 days of incubation at 37°C, the growth of the bacteria was recorded. The results presented are representative of at least three independent experiments. (B) (Left panel) Wild-type130b with pMMB2002 (⋄) or plbtB (⧫) and lbtB mutant NU303 with pMMB2002 (▵) or plbtB (▴) were inoculated into deferrated CDM containing 2 mM IPTG and then assayed for CAS reactivity as described in (A). The CAS reactivity of the mutant's cultures was significantly different from that of the wild-type and complemented mutant cultures, until 44 h (P < 0.05; Student's t test). The results presented are representative of at least four independent experiments. (Right panel) A <3-kDa supernatant fraction from the lbtB mutant and the complemented mutant NU303(plbtB) were tested in the feoB bioassay as indicated in A, with the results being representative of three independent experiments.

FIG. 6.

Amino acid sequence alignments of L. pneumophila LbtB with E. coli Bcr and TetA. The consensus sequences for conserved motifs A, B, C, D, G are labeled above the boxed-in areas; x is any amino acid, upper case is a highly conserved amino acid, and lower case is a conserved, but variable amino acid. Motif A, conserved in both 12- and 14-TMS families, is located in the cytoplasmic loop between TMS 2 and TMS 3 and may be involved in substrate binding as well as opening and closing of the channel (54). Motif B, located in TMS 4, is predicted to be involved in proton transfer (55). Motif C, located in TMS 5, is implicated in the direction of transport and is only found in those transport proteins with efflux capacity (30, 55). The function of motif D, located in TMS 1 in 12- and 14- TMS families, has not been investigated. Motif G, located in TMS 11, is found only in 12-TMS families, although its function is unknown (55).

FIG. 7.

Phenotypes of lbt mutant bacteria in the feoB bioassay. 10⁵ CFU of feoB mutant bacteria were plated unto the surface of non-iron-supplemented BCYE agar containing 2 mM IPTG, and then 130b, lbtA mutant NU300, or lbtB mutant NU303 containing either vector pMMB2002, plbtA, or plbtB was spotted on top of the agar as indicated. After 5 days of incubation at 37°C, the growth of the bacteria was recorded. The results presented are representative of three independent experiments.

FIG. 8.

Growth of wild-type and lbtA mutant L. pneumophila in deferrated CDM containing citrate. (A) 130b with pMMB2002 (•) and NU302 with pMMB2002 (○) or plbtA (▾) were grown in BYE to an OD₆₆₀ of 1.0, inoculated into deferrated CDM with 1 mM citrate to an OD₆₆₀ of 0.3, and then incubated at 37°C. At various times, the growth of the cultures was monitored spectrophotometrically. The growth of NU302 was different from that of the wild type and complemented mutant from 8 to 44 h (P < 0.05; Student's t test). (B) L. pneumophila strains were grown in deferrated CDM containing the indicated amounts of citrate, and then, at 44 h, the CAS reactivity of supernatants from 130b (black bars), NU302 (striped bars), and NU302(plbtA) (gray bars) was examined. The values represent the means and standard deviations from duplicate cultures. The CAS reactivity of NU302 was significantly different from that of the wild type and complemented mutant in 0 and 0.4 mM citrate-containing cultures (P < 0.05; Student's t test). When the citrate-associated CAS activity was normalized across cultures, more CAS reactivity was detected in citrate-containing NU302 supernatants than in non-citrate-containing NU302 supernatants (P < 0.05; Student's t test). The results presented in this figure are representative of two independent experiments.