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PHYSIOLOGY AND METABOLISM

Regulation of Fructose Transport and Its Effect on Fructose Toxicity in Anabaena spp.

Justin L. Ungerer, Brenda S. Pratte, Teresa Thiel
Justin L. Ungerer
Department of Biology, University of Missouri—St. Louis, Research 223, St. Louis, Missouri 63121
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Brenda S. Pratte
Department of Biology, University of Missouri—St. Louis, Research 223, St. Louis, Missouri 63121
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Teresa Thiel
Department of Biology, University of Missouri—St. Louis, Research 223, St. Louis, Missouri 63121
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  • For correspondence: thiel@umsl.edu
DOI: 10.1128/JB.00886-08
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  • FIG. 1.
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    FIG. 1.

    Fructose transport genes. (A) The region of the chromosome of A. variabilis with the fructose transport genes, namely, frtR (ava2170), encoding a putative lacI-like transcriptional regulator, and frtABC (ava2171 to -2173), encoding a putative periplasmic binding component, ATPase component, and transmembrane component, respectively. (B) Alignment of the promoter region of hrmE of N. punctiforme with a conserved region of the frtA promoter region, beginning about 300 bp upstream from the start codon of frtA. The transcription start site of hrmE is indicated by an arrow, and the −10 and −35 regions of the hrmE promoter are labeled. The HrmR binding sites, which are underlined, are shown within boxes that indicate longer conserved palindromic sequences.

  • FIG. 2.
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    FIG. 2.

    Growth of strains with or without fructose transport genes. Cells of A. variabilis strain FD or Anabaena sp. strain PCC 7120 with or without ftrRABC genes were grown on BG-11 agar medium with or without 5 mM fructose (F) for 4 days in the light or 7 days in the dark. Lane 1, Anabaena sp. strain PCC 7120 BP291, containing the frtRABC genes; lane 2, Anabaena sp. strain PCC 7120 BP292, containing the frtABC genes; lane 3, A. variabilis BP301 (frtR mutant); lane 4, Anabaena sp. strain PCC 7120; lane 5, A. variabilis strain FD.

  • FIG. 3.
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    FIG. 3.

    Transcription of frtA, frtR, hrmR, and hrmE. (A) Transcription of frtA and frtR was determined by RT-PCR, using RNAs extracted from A. variabilis strains grown with or without 5 mM fructose for 24 h. Lanes 1 and 2, wild-type A. variabilis; lanes 3 and 4, BP291 (Anabaena sp. strain PCC 7120 with frtRABC); lanes 5 and 6, BP292 (Anabaena sp. strain PCC 7120 with only frtABC); lanes 7 and 8, BP301 (A. variabilis frtR mutant); lanes 9 and 10, BP356 (A. variabilis strain overexpressing frtR); lane 11, positive control (FD DNA). Transcription of rnpB was the control for equal amounts of RNA in each reaction. (B) Transcription of hrmR and hrmE was determined by RT-PCR, using RNAs extracted from N. punctiforme grown in AA/8 without (−F) or with (+F) 5 mM fructose for 24 h. Con, positive control using chromosomal DNA from N. punctiforme. 16S rRNA was the control for equal amounts of RNA in each reaction.

  • FIG. 4.
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    FIG. 4.

    Expression of frtA-lacZ and frtR-lacZ fusions. (A) Relative rates of β-galactosidase activity were measured in strains BP352 (lacZ inserted within the frtA gene), JU338 (containing a 500-bp frtA promoter fragment fused to lacZ), BP353 (lacZ inserted within the frtR gene), and JU336 (containing a 400-bp frtR promoter fragment fused to lacZ), grown in AA/8 with or without 5 mM fructose. (B) Relative rates of β-galactosidase activity were measured in strains JU355 (containing a 500-bp frtA promoter fragment fused to lacZ in strain BP301, the frtR mutant), JU357 (containing a 500-bp frtA promoter fragment fused to lacZ in Anabaena sp. strain PCC 7120), JU353 (containing a 400-bp frtR promoter fragment fused to lacZ in strain BP301, the frtR mutant), and JU356 (containing a 400-bp frtR promoter fragment fused to lacZ in Anabaena sp. strain PCC 7120), grown with or without 5 mM fructose.

  • FIG. 5.
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    FIG. 5.

    Binding of FrtR to the promoter region of frtA. A 32P-labeled 131-bp DNA fragment upstream of frtA was incubated with or without recombinant FrtR protein. Samples in lanes 2 and 3 contained 100 ng and 300 ng of FrtR protein extract, respectively. Samples in lanes 4 to 7 contained 700 ng of FrtR protein extract.

  • FIG. 6.
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    FIG. 6.

    Growth of strains with fructose. The strains indicated for each panel were grown in AA/8 without fructose and then diluted in medium containing the concentrations of fructose indicated by the symbols on day 0. (A) Strains FD (A. variabilis wild type) (solid symbols) and BP301 (frtR mutant) (open symbols). (B) Wild-type strain FD. (C) A. variabilis BP301 (frtR mutant). (D) Anabaena sp. strain PCC 7120 with the frtRABC genes (strain BP291). (E) Anabaena sp. strain PCC 7120 with the frtABC genes (lacking frtR) (strain BP292). (F) Strain JU377, a strain of A. variabilis in which the frtABC genes are constitutively expressed from the strong psbA promoter. (G) Wild-type strain FD grown in the dark. (H) Anabaena sp. strain PCC 7120 with the frtRABC genes (strain BP291) grown in the dark. Fructose concentrations were as follows: ▾, 0 mM; ▪, 1 mM; ⧫, 5 mM; +, 10 mM; •, 50 mM; and ▴, 200 mM.

  • FIG. 7.
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    FIG. 7.

    Fructose transport in wild-type (⧫) and BP301 (frtR mutant) (▪) strains. Cells were grown in AA/8 to an OD720 of 0.250, fructose was added at time zero, and transport was measured as the disappearance of fructose from the medium over time (hours [A] or minutes [B]). Fructose was measured using a fructose assay kit (Sigma-Aldrich).

  • FIG. 8.
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    FIG. 8.

    Light micrographs of filaments of Anabaena sp. strain PCC 7120 with the frtABC genes (strain BP291) grown without (A) or with (B) 5 mM fructose and of filaments of A. variabilis FD grown without (C) or with (D) 5 mM fructose. The size scale is the same for all panels. Heterocysts are indicated by arrows.

Tables

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  • TABLE 1.

    Strains and plasmids used for this study

    Strain or plasmidRelevant characteristicsReference
    Strains
        A. variabilis FD Anabaena variabilis parent strain 9
        Anabaena sp. strain PCC 7120Wild-type strain
        Anabaena sp. strain PCC 7120 BP291 frtRABC expressed from plasmid pBP291This work
        Anabaena sp. strain PCC 7120 BP292 frtABC expressed from plasmid pBP292This work
        A. variabilis FD BP301 frtR mutated with insert of Nmr cassette at NaeI sitesThis work
        A. variabilis BP352pBP352 integrated into the chromosomeThis work
        A. variabilis BP353pBP353 integrated into the chromosomeThis work
        A. variabilis BP356pBP356 integrated into the chromosomeThis work
        A. variabilis JU336pJU336 integrated into the chromosomeThis work
        A. variabilis JU338pJU338 integrated into the chromosome
        A. variabilis JU377pJU377 integrated into the chromosomeThis work
        Anabaena sp. strain PCC 7120 JU356pJU336 integrated into the chromosomeThis work
        Anabaena sp. strain PCC 7120 JU357pJU338 integrated into the chromosomeThis work
        Anabaena FD JU353pJU336 integrated into the chromosome of A. variabilis FD BP301This work
        Anabaena FD JU355pJU338 integrated into the chromosome of A. variabilis FD BP301This work
    Plasmids
        pAAWY30099.3-kb clone of A. variabilis DNA containing frtRABC; Cmr JGI sequencing clone
        pBP285Kmr Nmr cassette in a polylinker [C.K.3 (10)] with a transcriptional terminatorThis work
        pBP288Cloning vector for integration of transcriptional fusions into the chromosome; Tcr Kmr Nmr Spr Smr Apr This work
        pBP2899.8-kb EcoRI fragment of pAAWY3009 (containing Ava2169 to Ava2173 but lacking Ava4074), self-ligatedThis work
        pBP2918.4-kb ScaI/SmaI fragment from pBP289 (containing frtRABC) inserted into the SmaI sites of pRL57This work
        pBP2924.9-kb NaeI/SmaI fragment from pBP289 (containing frtABC) ligated into the SmaI sites of pRL57This work
        pBP2991.1-kb SmaI fragment containing the Nmr cassette of pBP285 inserted into NaeI site of frtR in pHL110This work
        pBP3015-kb BglII fragment of pRL1075 ligated into the BamHI site of pBP299This work
        pBP313Cloning vector to overexpress genes from psbA promoter; Kmr Nmr Spr Smr Apr This work
        pBP314PCR-amplified Tcr cassette of pBR322 inserted into the NdeI/BamHI sites of pET22bThis work
        pBP328PCR-amplified Tcr cassette of pBR322 inserted into the SmaI/SacI sites of pBP313This work
        pBP350Replaced BglII/BamHI fragment containing Nmr cassette of pPE20 with PCR-amplified Ω Spr Smr cassette from pBP288This work
        pBP3514.2-kb EcoRI/ScaI fragment of pHL110 (containing Ava2169 to -71) cloned into EcoRV/EcoRI fragment of pBR322This work
        pBP3525-kb SmaI fragment (containing a lacZ Spr Smr cassette) of pBP350 inserted into EcoRV site of pBP351This work
        pBP3535-kb SmaI fragment (containing a lacZ Spr Smr cassette) of pBP350 inserted into ClaI site (blunted) of pBP351This work
        pBP354PCR-amplified frtR gene inserted into NdeI/BamHI sites of pBP314 for overexpression of native FrtRThis work
        pBP356PCR-amplified frtR gene inserted into SmaI/SacI sites of pBP313 under the control of psbA promoterThis work
        pBR322Mobilizable plasmid; Apr Tcr 5
        pET22bT7 expression vector; expression induced by isopropyl-β-d-thiogalactopyranosideInvitrogen
        pHL1104.5-kb HindIII frt region of pAAWY3009 inserted into HindIII site of pUC18This work
        pJU336PCR-amplified 400-bp frtR promoter fragment inserted into BglII/SmaI sites of pBP288This work
        pJU338PCR-amplified 500-bp frtA promoter fragment inserted into BglII/SmaI sites of pBP288This work
        pJU377PCR-amplified frtA gene inserted into SmaI/SacI sites of pBP313 under the control of psbA promoterThis work
        pPE20Source of lacZ for transcriptional fusions; Apr Kmr Nmr 44
        pRL1075Source of mobilization site, oriT, and sacB gene, which confers sucrose sensitivity; Cmr Emr 4
        pRL57S.K5 + L.HEH2 + C.S3; positive selection shuttle (pDU1) cloning vector 10
        pRL648Kmr Nmr cassette in a polylinker (C.K.3) 10
        pUC18Cloning vector; Apr 47
        pUC1819RICloning vector; Apr 7

Additional Files

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    • Supplemental file 1 - Table S1, primers.
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Regulation of Fructose Transport and Its Effect on Fructose Toxicity in Anabaena spp.
Justin L. Ungerer, Brenda S. Pratte, Teresa Thiel
Journal of Bacteriology Nov 2008, 190 (24) 8115-8125; DOI: 10.1128/JB.00886-08

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Regulation of Fructose Transport and Its Effect on Fructose Toxicity in Anabaena spp.
Justin L. Ungerer, Brenda S. Pratte, Teresa Thiel
Journal of Bacteriology Nov 2008, 190 (24) 8115-8125; DOI: 10.1128/JB.00886-08
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KEYWORDS

ATP-binding cassette transporters
Anabaena variabilis
Bacterial Proteins
Fructose

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