Proteobactin and a yersiniabactin-related siderophore mediate iron acquisition in Proteus mirabilis - PubMed (original) (raw)

Stephanie D Himpsl et al. Mol Microbiol. 2010 Oct.

Abstract

Proteus mirabilis causes complicated urinary tract infections (UTIs). While the urinary tract is an iron-limiting environment, iron acquisition remains poorly characterized for this uropathogen. Microarray analysis of P. mirabilis HI4320 cultured under iron limitation identified 45 significantly upregulated genes (P ≤ 0.05) that represent 21 putative iron-regulated systems. Two gene clusters, PMI0229-0239 and PMI2596-2605, encode putative siderophore systems. PMI0229-0239 encodes a non-ribosomal peptide synthetase-independent siderophore system for producing a novel siderophore, proteobactin. PMI2596-2605 are contained within the high-pathogenicity island, originally described in Yersinia pestis, and encodes proteins with apparent homology and organization to those involved in yersiniabactin production and uptake. Cross-feeding and biochemical analysis shows that P. mirabilis is unable to utilize or produce yersiniabactin, suggesting that this yersiniabactin-related locus is functionally distinct. Only disruption of both systems resulted in an in vitro iron-chelating defect; demonstrating production and iron-chelating activity for both siderophores. These findings clearly show that proteobactin and the yersiniabactin-related siderophore function as iron acquisition systems. Despite the activity of both siderophores, only mutants lacking the yersiniabactin-related siderophore have reduced fitness in vivo. The fitness requirement for the yersiniabactin-related siderophore during UTI shows, for the first time, the importance of siderophore production in vivo for P. mirabilis.

© 2010 Blackwell Publishing Ltd.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1. The growth rate of P. mirabilis HI4320 is decreased following culture in LB medium containing Desferal

P. mirabilis HI4320 cultured in LB medium containing 0, 15, 25, or 30 μM Desferal, an iron-chelating agent. A concentration of 15 uM Desferal in LB medium decreases the growth rate of wild-type in comparison to wild-type grown in LB medium alone.

Fig. 2

Fig. 2. qPCR of mRNA from P. mirabilis HI4320 cultured in LB medium containing 15 μM Desferal validates microarray results that identified up- and down-regulated genes in response to iron limitation

Log2 fold-change was determined relative to strain HI4320 cultured in LB medium alone. (A) qPCR validation of select HI4320 genes up- or down-regulated during iron limitation in vitro as determined by microarray. (B) qPCR validation of HI4320 putative yersiniabactin-related siderophore, nrp (PMI2596-2605) that was up-regulated during iron limitation in vitro as determined by microarray. (C) qPCR validation of HI4320 putative siderophore system proteobactin, pbt (PMI0229-0239) that was up-regulated during iron limitation in vitro as determined by microarray (black bars) and down-regulated following the addition of 25 μM FeCl3·6H2O to an iron-chelated LB medium culture of bacteria (grey bars). (D) qPCR confirmation of HI4320 siderophore biosynthesis genes pbtA (PMI0232) and nrpR (PMI2599) expression in vivo. mRNA, isolated from bacteria recovered from the urine of experimentally infected CBA/J mice, was subjected to qPCR. A significant difference in expression levels (*, P = 0.0002) was determined using a two-tailed unpaired _t_-test.

Fig. 3

Fig. 3. Transcriptional organization of putative siderophore system PMI2596-2605 and PMI0229-0239 in P. mirabilis HI4320

(A, C) mRNA for cDNA synthesis was isolated from wild-type HI4320 following culture in LB medium containing15 μM Desferal. For PCR reactions: +, reverse transcriptase added to cDNA synthesis reaction; −, no reverse transcriptase added, mRNA used as template; G, HI4320 genomic DNA used as template. (B, D) Siderophore biosynthesis and transport gene clusters of HI4320 drawn to scale with primer design for RT-PCR operon mapping. Thick arrows represent open reading frames that indicate direction of transcription. Ferric uptake regulator (Fur) recognition sequences were identified based on homology to those of P. aeruginosa using a promoter analysis program in the Virtual Footprint software suite (Munch et al., 2005). Location and direction of Fur recognition sequences are indicated. Intergenic primer pairs used for RT-PCR are represented by alphabetical letters. Thin solid arrows represent direction and span the length of genes that are required to make a complete transcript. Proposed gene nomenclature for transcripts are indicated below thin solid lines and size of transcript are indicated above the solid lines.

Fig. 4

Fig. 4. The yersiniabactin-related siderophore and proteobactin of P. mirabilis are similar to siderophore biosynthesis and transport gene clusters in other Enterobacteriaceae as shown by gene sequence alignments

(A) P. mirabilis yersiniabactin-related siderophore is homologous to that of P. syringae DC3000 and Y. pestis KIM. The yersiniabactin-related siderophore of P. mirabilis lacks the genes involved in salicyclic acid biosynthesis and incorporation, ybtS and ybtE of Y. pestis and irp5, pchB, and pchA of P. syringae. The nrp locus of P. mirabilis also lacks a transcriptional regulator but encodes a NRPS-associated methyl-transferase and a phosphopantetheinyl-transferase which are not located within the yersiniabactin operons of P. syringae and Y. pestis. (B) The newly identified NIS system of P. mirabilis, proteobactin, is similar to identical gene clusters found in P. carotovora subsp. atroseptica (shown) and P. carotovora subsp. carotovora.

Fig. 5

Fig. 5. P. mirabilis is unable to utilize yersiniabactin produced by uropathogenic E. coli 536

LB medium was supplemented with 25 μM Desferal, the minimum concentration of chelator required to completely suppress growth of P. mirabilis. Lactose-fermenting E. coli are distinguished from P. mirabilis by the using the chromogenic indicator X-gal. (A) Growth of wild-type P. mirabilis, the proteobactin synthesis/receptor mutant (PbtSR), and the yersiniabactin-related siderophore/receptor mutant (NrpSR) can be restored when cross-fed by E. coli 536 that produces both enterobactin and yersiniabactin. (B, C) Restoration of P. mirabilis growth for all strains tested is abolished when cross-fed by the either E. coli 536 enterobactin mutant, entF::kan, or the enterobactin/yersiniabactin double mutant, entF ybtS::kan. (D) P. mirabilis growth can be restored using E. coli CFT073, an enterobactin producing strain incapable of synthesizing yersiniabactin.

Fig. 6

Fig. 6. Preparative HPLC and LC-MS analysis demonstrates P. mirabilis HI4230 is incapable of producing yersiniabactin

Preparative HPLC chromatogram (upper traces) performed on crude supernatant extracts from yersiniabactin-producing E. coli 536 entF::kan (dashed line) and yersiniabactin-deficient entF ybtS::kan (solid line) cultures to demonstrate production/lack of production of yersiniabactin. The lower traces represent chromatograms using the same HPLC conditions from P. mirabilis HI4230 pbtA::kan (dashed line) and nrpR pbtA::kan (solid line). The y-axis represents arbitrary units (AU) to offset sample traces. The inset indicates the LC-MS (positive ion mode) analysis of E. coli yersiniabactin present in the dominant peak (*) isolated at 15 min by HPLC. Iron-bound yersiniabactin is known to have an ion intensity at m/z of 535.1.

Fig. 7

Fig. 7. Phylogenetic analysis of 92 homologous protein sequences to NIS synthetase PbtA involved in the assembly of proteobactin

The NIS synthetase enzymes are split into three subfamilies A, B, and C. Proteobactin synthetase, PbtA, (boxed) is placed with the type B subfamily clade of NIS synthetases which includes Y4xN, a siderophore synthetase previously found to be homologous to other NIS synthetases of this group.

Fig. 8

Fig. 8. Siderophore production by P. mirabilis HI4320

(A) Detectable siderophore production on chrome azurol S (CAS) agar of overnight LB bacterial cultures spotted in 5 μl volumes. Blue to orange color change of agar indicates siderophore production. (B) CAS assay depicting siderophore production from filtered bacterial supernatants following culture in MOPS defined medium with (black bars) and without (white bars) 0.1 mM FeCl3·6H2O. Only supernatants of P. mirabilis samples were concentrated 50-fold. Supernatant of E. coli CFT073 and double mutant, entF::kan iucB::cam, were not concentrated. Positive control of phosphate buffered saline containing 15 μM Desferal (grey bar). A lower absorbance (A630) indicates greater siderophore production. Significant differences in absorbance (*, P ≤ 0.0022) was determined using a two-tailed unpaired _t_-test.

Fig. 9

Fig. 9. A proposed model for iron acquisition systems in P. mirabilis

Hypothetical functions of the proteins listed are based on up-regulation of their genes under iron limitation, homology with genes from other bacterial species, and functional studies conducted in this report and related bacterial species. Outer membrane (OM) receptors are dependent on the energy transducing complex TonB-ExbB-ExbD (shown in red) for intracellular transport of iron. Heme uptake systems are displayed in green. Heme uptake may also be carried out by the outer membrane receptor, HemC. Import of periplasmic ferrous iron is performed by the ABC transporter SitABCD (shown in yellow) located on the inner membrane (IM). A putative ferric citrate system (shown in orange) encodes a TonB-dependent receptor PMI3706, and accessory proteins. Proteins involved in siderophore biosynthesis and transport of ferri-siderophores are shown in blue. Ferri-siderophores are transported by outer membrane receptors IreA, FyuA, FatA, and FhuA. Ferri-siderophores transported to the periplasm are shuttled to inner membrane transport proteins, NrpAB and PMI0229-0230. Additional putative ferri-siderophore transport proteins include the periplasmic binding protein, CeuE, and inner membrane transport proteins, PMI2957-2960. Cellular proteins, Aad, PMI2149, ViuB and, may be involved in siderophore synthesis and the regulation and utilization of iron of ferri-siderophore. Putative TonB-dependent receptors possibly involved in colicin uptake are shown in purple. Cytoplasmic iron-containing proteins include nrdEFIH and sufABCDSE (shown in white). Bfd, an iron removal protein, and PMI2503 involved in iron metabolism are shown in brown.

References

    1. Alteri CJ, Mobley HL. Quantitative profile of the uropathogenic Escherichia coli outer membrane proteome during growth in human urine. Infection and immunity. 2007;75:2679–2688. -PMC -PubMed
    1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of molecular biology. 1990;215:403–410. -PubMed
    1. Andrews SC, Robinson AK, Rodriguez-Quinones F. Bacterial iron homeostasis. FEMS microbiology reviews. 2003;27:215–237. -PubMed
    1. Berger H, Hacker J, Juarez A, Hughes C, Goebel W. Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli. Journal of bacteriology. 1982;152:1241–1247. -PMC -PubMed
    1. Braun V. Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins. FEMS microbiology reviews. 1995;16:295–307. -PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources