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Molecular Pathogenesis

Identification of Francisella tularensis Genes Affected by Iron Limitation

Kaiping Deng, Robert J. Blick, Wei Liu, Eric J. Hansen
Kaiping Deng
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9048
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Robert J. Blick
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9048
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Wei Liu
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9048
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Eric J. Hansen
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9048
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  • For correspondence: eric.hansen@utsouthwestern.edu
DOI: 10.1128/IAI.01975-05
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  • FIG. 1.
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    FIG. 1.

    Growth of F. tularensis LVS under iron-replete and iron-restricted conditions. F. tularensis LVS was grown overnight on an MH+ agar plate. For iron-replete growth (filled circles), bacteria from the plate were inoculated into 1 ml of MH+ broth and grown at 37°C overnight. This overnight culture was then used to inoculate 10 ml MH+ broth to an OD600 of 0.05. For iron-restricted growth, the overnight culture was obtained by inoculating the bacteria from the plate into 1 ml MH− broth. This overnight culture was used to inoculate 20 ml of MH−/DF broth. When the culture reached an OD600 of 0.4, the 20-ml MH−/DF culture was divided into two cultures of 10 ml each. Fe-PPi was added to a final concentration of 330 μM in one culture (filled triangles), whereas an equivalent volume of sterile H2O was added to the other culture (open triangles).

  • FIG. 2.
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    FIG. 2.

    Identification of F. tularensis LVS proteins with increased expression under iron-restricted conditions. F. tularensis LVS cells were grown to stationary phase under both iron-replete (lane 1) and iron-restricted (lane 2) conditions. Cell envelopes and whole-cell lysates were prepared as described in Materials and Methods. (A) Proteins in cell envelopes were resolved by SDS-PAGE and stained with Coomassie blue. Molecular mass standards are present in the far left lane. The black arrow indicates the IglC protein, and the white arrow indicates the PdpB protein. Proteins in cell envelopes (B) or in whole-cell lysates (C) were resolved by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and probed in a Western blot analysis with polyclonal mouse antiserum to PdpB (B) or FigA (C).

  • FIG. 3.
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    FIG. 3.

    Putative Fur boxes associated with the F. tularensis Schu4 pdpB, iglC, and figA ORFs. The ClustalW alignment was accomplished with the MacVector 6.5.3 software. The consensus Fur box is outlined. Identical bases in each pair are underlined and bold; nonidentity is indicated by italics.

  • FIG. 4.
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    FIG. 4.

    F. tularensis Schu4 figA and adjacent ORFs. (A) Schematic drawing. (B) RT-PCR analysis of possible transcriptional linkage between figA and adjacent ORFs. RT-PCR was carried out as described in Materials and Methods, with oligonucleotide primer pairs spanning intergenic regions 1 (5′-GTGCTGAAATGATTGACTATAGTCTC-3′ and 5′-CTTGTCATTGTTTATATTGTAGAAGAATA-3′), 2 (5′-AGTGCGATAAAGATGACATGC-3′ and 5′-TAGCTGCTATGATTATAAGC-3′), 3 (5′-TATCTATTTGACCTTAAATC-3′ and 5′-TGAATAATTGTTTCAATTTG-3′), and 4 (5′-TCAATACCAAGTTCTCAGAG-3′ and 5′-ACCTAAAAACCATGTTAAAAG-3′), respectively. Lanes 1, 4, 7, and 10, PCR products derived from F. tularensis LVS chromosomal DNA. The sizes of these PCR products are 547 bp (lane 1), 135 bp (lane 4), 120 bp (lane 7), and 121 bp (lane 10). Lanes 2, 5, 8, and 11, RT-PCR negative controls lacking reverse transcriptase. Lanes 3, 6, 9, and 12, products obtained when these same primers were used in an RT-PCR with F. tularensis LVS total RNA. Size markers are present on the left side of panel B.

  • FIG. 5.
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    FIG. 5.

    Construction and characterization of figA mutants. (A) Schematic of PCRs used to amplify and sew flanking regions of the F. tularensis LVS figA gene. The promoterless kan cartridge was ligated into this cloned PCR product at the KpnI site. (B) PCR products obtained from the chromosomal DNAs of wild-type and figA mutant strains with oligonucleotide primers (described in Results) binding 1.5 kb from the 5′ and 3′ ends of the figA gene. Lane 1, wild-type F. tularensis LVS; lane 2, F. tularensis LVS figA::np-kan; lane 3, wild-type F. novicida U112; lane 4, F. novicida U112 figA::np-kan. The positions of 4.0-kb and 5.0-kb size markers are indicated on the left. (C) PCR products obtained from chromosomal DNAs of wild-type and mutant strains with primers binding within the kan cartridge; lanes are the same as in panel B. The position of an 850-bp size marker is shown on the left. (D) Western blot analysis of whole-cell lysates of these wild-type and mutant strains grown in MH−/DF broth. Lanes 1 to 4 contain whole-cell lysates of the same strains as in panel B; lane 5 contains a whole-cell lysate of F. novicida U112 figA::np-kan (pKD107). The primary antibody was mouse FigA antiserum. (E) Western blot analysis of these same lysates with rat polyclonal F. tularensis LVS FopA antiserum (i.e., a loading control).

  • FIG. 6.
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    FIG. 6.

    CAS assay. Wild-type F. tularensis LVS and F. novicida U112 and mutant and complemented mutant strains of both were grown on CAS/CDM− agar plates. (A) Wild-type F. tularensis LVS (sector 1) and F. tularensis LVS figA::np-kan (sector 2) grown for 5 days. (B) Wild-type F. novicida U112 (sector 1), F. novicida U112 figA::np-kan (sector 2), F. novicida U112 figA::np-kan(pKD107) (sector 3), F. novicida U112 figA::np-kan(pFNLTP-CAT) (sector 4), and F. novicida U112 figA::np-kan(pKD108) (sector 5) grown for 24 h.

  • FIG. 7.
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    FIG. 7.

    Extracellular and intracellular growth of wild-type and mutant strains of F. tularensis LVS and F. novicida U112. For extracellular growth (A and B), bacteria were grown in both iron-replete (filled symbols) and iron-restricted (open symbols) media. The wild-type F. tularensis LVS strain (panel A, circles), its figA mutant (A, triangles), the wild-type F. novicida strain (B, circles), and its figA mutant (B, triangles) were grown as described in Materials and Methods. To assess intracellular growth (C and D), these same strains were grown in the macrophage cell line J774A.1 as described in Materials and Methods. (C) Wild-type F. tularensis LVS (filled columns) and its figA mutant (open columns). (D) Wild-type F. novicida (filled columns) and its figA mutant (open columns). These data are the means from two independent experiments. In panel D, the difference between the F. novicida wild-type and mutant strains was significant only at 24 h postinfection (asterisk; P = 0.0096, Student's t test).

  • FIG. 8.
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    FIG. 8.

    Cross-feeding of the F. novicida figA mutant by the F. novicida U112 wild-type (w.t.) strain. The F. novicida U112 wild-type strain (streak 1) and its figA mutant (streaks 2, 3, and 4) were inoculated onto both CAS/CDM− (A) and CDM+ (B) agar plates and grown for 36 h.

Tables

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  • TABLE 1.

    Bacterial strains and plasmids used in this study

    Strain or plasmidGenotype or descriptionReference or source
    E. coli strains
        DH5αHost strain for cloning experiments 34
        HB101Host strain essential for propagating plasmids carrying mutated Francisella genes used for electroporation of Francisella 34
    Francisella tularensis strains
        LVS no. 11 F. tularensis LVS 10
        LVS figA::np-kanMutant of LVS with promoterless kan cartridge inserted in figA geneThis study
        LVS figA::np-kan(pKD107)LVS figA::np-kan containing pKD107 with F. novicida U112 figA gene; expresses FigA proteinThis study
    Francisella novicida strains
        U112Wild-type strain of F. novicida 26
        U112 figA::np-kanMutant of U112 with promoterless kan cartridge inserted in figA geneThis study
        U112 figA::np-kan(pKD107)U112 figA::np-kan containing pKD107 with F. novicida U112 figA gene; expresses FigA proteinThis study
        U112 figA::np-kan(pKD108)U112 figA::np-kan containing pKD108 with F. tularensis LVS figA gene; expresses FigA proteinThis study
        U112 figA::np-kan(pFNLTP-CAT)U112 figA::np-kan containing vector pFNLTP-CATThis study
    Plasmids
        pACYC184Broad-host-range cloning vector; Chlorr TetrNew England Biolabs
        pKD101pACYC184 with 2.2-kb fragment containing figA flanking regionsThis study
        pKD101-np-kanpKD101 with promoterless kan cartridge inserted into KpnI siteThis study
        pFNLTP6Shuttle vector for Francisella; Kanr Ampr 24
        pKD105pFNLTP6 with F. tularensis LVS figA gene inserted into BamHI siteThis study
        pKD108pKD105 with ΔEcat cartridge inserted into XhoI site; this vector backbone containing the ΔEcat cartridge is the same as that of pFNLTP-CATThis study
        pFNLTP-CATpFNLTP6 with ΔEcat cartridge inserted in XhoI site; Kanr Ampr ChlorrThis study
        pKD107pFNLTP-CAT carrying F. novicida U112 figA geneThis study
  • TABLE 2.

    F. tularensis LVS genes whose expression was maximally altered by growth under iron-restricted conditionsa

    F. tularensis Schu4 ORF no.Gene identity or functionAvg fold differenceb
    Genes up-regulated under iron-restricted growth conditions
        29cHypothetical protein (FigA)19.70
        27cDiaminopimelate decarboxylase (FigC)15.13
        26cHypothetical protein (FigD)14.78
        28cHypothetical protein (FigB)9.78
        1565cGlycosyl hydrolase, family 3, pseudogene4.08
        1702Conserved hypothetical protein3.87
        1701Hypothetical protein3.62
        1707Conserved hypothetical protein3.41
        1542cOuter membrane protein3.29
        1714cIntracellular growth locus, subunit A (IglA)3.26
        383Hypothetical protein3.22
        989Hypothetical protein3.19
        1709Conserved hypothetical protein3.09
        980Aminotransferase, class II3.08
        1711cIntracellular growth locus, subunit D (IglD)3.06
        1703Conserved hypothetical protein3.01
        1700Conserved hypothetical protein (PdpB)2.99
        1706Conserved hypothetical protein2.99
        1717Major facilitator superfamily transport protein2.98
        1712cIntracellular growth locus, subunit C (IglC)2.96
    Genes down-regulated under iron-restricted growth conditions
        1214cHaloacid dehalogenase-like hydrolase family protein0.32
        881cAmino acid permease0.39
        265ABC transporter, membrane protein0.42
        1269cChaperone protein, DnaK (heat shock protein family 70)0.43
        1696Chaperone protein, GroEL0.43
        1624cHypothetical protein0.44
        1695Chaperone protein GroES0.44
        938Adenosylmethionine-8-amino-7-oxononanoate aminotransferase0.46
        33130S ribosomal protein S30.47
        172Hypothetical membrane protein, fragment0.48
        32750S ribosomal protein L230.49
        413c1,4-α-Glucan branching enzyme0.49
        32850S ribosomal protein L20.49
        14050S ribosomal protein L110.50
        32650S ribosomal protein L40.50
        1048cHypothetical protein0.51
        266ABC transporter, ATP-binding protein0.51
        551Conserved hypothetical protein, pseudogene0.52
        999cZIP metal transporter family protein, pseudogene0.52
        33930S ribosomal protein S80.52
    • ↵ a For detailed DNA microarray data, see Table S1 in the supplemental material.

    • ↵ b The average fold difference indicates the gene expression level under iron-restricted conditions relative to that obtained under iron-replete conditions.

  • TABLE 3.

    qRT-PCR analysis of gene expressiona

    Schu4 ORF no.Putative functionFold differencebqRT-PCR primer pair (5′-3′)d
    MicroarrayqRT-PCR
    26cHypothetical protein (FigD)14.813.4F: AAGCCTAATGGTAGCTGGTGAATC
    R: TTTTGGTGAGACTCCGTAAGTTTTT
    29cHypothetical protein (FigA)19.724.7F: GGCTACTAAAGACAAAACACTAATGCC
    R: CAGACTCAGGCGATACTGTTCTTG
    27cDiaminopimelate decarboxylase (FigC)15.113.0F: GGTTTAGCAGGAATAAGCCCTACTC
    R: TTGTCGCGGCATATAGTTCTAAATC
    28cHypothetical protein (FigB)9.919.1F: GAGTTTTTTAGCTCAGACCCGATAATC
    R: ATCGGCGCTGAAAATAGCA
    651cProton-dependent oligopeptide family protein2.40.6F: CTCGCAAAAAAGACATTAGTATAAAATTAAAT
    R: AAGTTGCAAATTGTCCCCAAA
    1712cIntracellular growth locus, subunit c (IglC)3.02.6F: AAAAAGGAGAATGATTATGAGTGAGATG
    R: TGCAGTAGGATCAGTTCTCACATG
    403Peptide deformylase2.83.2F: CAGCTATAATATTTCAGCATGAATTTAATCA
    R: AGCAAATTTAGCTTGTAGTTCTTCGTT
    1699Conserved hypothetical protein (PdpA)2.62.6F: TGAGTTAATTTCAAACTCTGCCATATC
    R: GTTTGGGTATATGCCATTTCACAG
    1780Putative transposase1.51.1F: AAGAACCGGAGATTATAGTTCAAAGC
    R: AAATACTGTTCAATCAATGTTTTATCGG
    651cProton-dependent oligopeptide family protein1.30.7F: CGTTGATAGTAAAACTATTGGTTTTGCA
    R: GGAAGAGCAACCACTTGATTTAATAGA
    664cHistidine decarboxylase1.01.1F: CATTTGATGGTGCTTTTCTACCG
    R: ATATTCCTGAAGGCATCGGATTAC
    583Outer membrane-associated protein0.91.0F: CAAATCTAGCAGGTCAAGCAACAG
    R: AACACTTGCTTGAACATTTCTAGATAGTTC
    700Conserved hypothetical protein0.80.6F: AAAATGGTGTTCGCCTTTTGG
    R: CCTTCGACATCATCTTTGTAGTCAAC
    445ABC transporter, ATP-binding, pseudogene0.91.2F: TTAGAAGAGTTTGTTGGAGGATATGATG
    R: ATTTTTCGACAACTCACTGTTATTTTTAGT
    881cAmino acid permease0.40.6F: CCAAGGTTCAGAAATTATCGGACTA
    R: GTGCTTAGCGATTCTTGGCAT
    50Translation initiation factor IF20.60.8F: TTGGCGCGTATTCTGTTAAGAC
    R: GCTCGCATTGAAGTAAAAGCCT
    14050S ribosomal protein L110.50.8F: ACGCCACCTGCTTCTTACTTAATT
    R: CGCACGAGTTATAGTTCCAACAA
    350DNA-directed RNA polymerase, alpha subunit0.70.7F: TTGACAAACCAAAGAGCATTTAGC
    R: TCTCAAGCTCGGTTGGTATCG
    1695Chaperone protein, GroES0.40.5F: GCTCAAGAGAAACCTAGCCAAGG
    R: ACATCCATAGGTAGCGTAGTGCC
    32650S ribosomal protein L40.50.7F: AGTTTCTGGTGGCGGTGC
    R: GGTGAACGGATAGTACCCGCT
    1696Chaperone protein, GroEL0.40.7F: TTCAAAGACAGCGGATGTTGC
    R: GCGACAGCTTTTAGACCCTCTG
    • ↵ a qRT-PCR for each gene was performed with two individually prepared RNA samples, each in triplicate.

    • ↵ b The fold difference indicates the gene expression level under iron-restricted conditions relative to that obtained under iron-replete conditions.

    • ↵ c ORF 651 in the F. tularensis Schu4 genome was found to be fragmented into two separate ORFs in the F. tularensis LVS genome by the deletion of a single nucleotide.

    • ↵ d F, forward; R, reverse.

Additional Files

  • Figures
  • Tables
  • HTML Page - index.htslp

    Files in this Data Supplement:

    • Supplemental file 1 - Table S1. DNA microarray results.
      Zipped Excel document, 190K.
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Identification of Francisella tularensis Genes Affected by Iron Limitation
Kaiping Deng, Robert J. Blick, Wei Liu, Eric J. Hansen
Infection and Immunity Jun 2006, 74 (7) 4224-4236; DOI: 10.1128/IAI.01975-05

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Identification of Francisella tularensis Genes Affected by Iron Limitation
Kaiping Deng, Robert J. Blick, Wei Liu, Eric J. Hansen
Infection and Immunity Jun 2006, 74 (7) 4224-4236; DOI: 10.1128/IAI.01975-05
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KEYWORDS

Bacterial Proteins
Francisella tularensis
Genes, Bacterial
iron

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