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Articles

Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

Michael S. Carter, Birgit E. Alber
J. P. Armitage, Editor
Michael S. Carter
Department of Microbiology, Ohio State University, Columbus, Ohio, USA
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Birgit E. Alber
Department of Microbiology, Ohio State University, Columbus, Ohio, USA
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J. P. Armitage
Roles: Editor
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DOI: 10.1128/JB.00402-15
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  • FIG 1
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    FIG 1

    Comparison of pccB transcript abundance (A) and the PCC activity (B) of R. sphaeroides 2.4.1 and RsΔpccRMC12 cells grown with succinate (filled symbols), acetate (open symbols), and propionate-HCO3− (hatched symbols). Expression levels were relative to the average value of recA and rpoZ transcript abundance. Error bars represent the standard deviations from at least three biological replicates. RsΔpccRMC12 did not grow with propionate-HCO3−.

  • FIG 2
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    FIG 2

    (A) Genomic context of pccB (encoding the β subunit of propionyl-CoA carboxylase), pccR, and the consensus sequence (open boxes) initially identified upstream of pccR genes within several alphaproteobacteria. Striped arrows, genes annotated to encode a multidrug-resistant transporter; checkered arrows, conserved coding regions of unknown function; open arrows, coding regions with no significant sequence identity to the other genes shown. (B) Illustration of the mutated pccB upstream regions that are present on each plasmid in the pMC85 series. Each plasmid contains a derivative of a 215-bp fragment of the pccB upstream fragment including 27 bp of the pccB coding region fused to lacZ. Regulation of expression from each putative promoter is presented in Table 2.

  • FIG 3
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    FIG 3

    Photoheterotrophic growth of R. sphaeroides 2.4.1 (open circles), RsΔpccRMC12 (filled circles), RsΔpccRMC12(pBBRsm2MCS5) (crosses), and RsΔpccRMC12(pMC66) (stars) with succinate, acetate, and propionate-HCO3−.

  • FIG 4
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    FIG 4

    Photoheterotrophic growth of R. sphaeroides 2.4.1 (left) and RsΔpccRMC12 (right) that carry no plasmid (circles), pMC85 (diamonds), pMC85Δ1 (triangles), pMC85Δ2 (inverted triangles), or pMC85Δ12 (squares) with succinate, acetate, or propionate-HCO3−.

  • FIG 5
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    FIG 5

    Neighbor-joining tree of selected ScfR proteins. Nodes with bootstrap values greater than 50% are labeled. Each entry includes the class name assigned to the given protein on the basis of the genomic context of its respective gene. A question mark indicates that the corresponding genetic context does not contain any genes of the indicated metabolic gene clusters. An asterisk indicates a protein that clusters in the tree with proteins of a different class. Following the protein name is the locus tag for the given protein and the species in which the protein is found. To the right of the tree is an illustration of the general genome neighborhood of the genes that encode proteins from the corresponding clade. See Fig. S2 in the supplemental material for the extended tree. A DNA logo (50) is included if a conserved motif was identified upstream of the genes responsible for the proteins in the corresponding clade. The indicated motifs may have occurred multiple times in the queried sequences. Gene annotations are as follows: pccB, propionyl-CoA carboxylase β subunit; pccA, propionyl-CoA carboxylase α subunit; mcm, methylmalonyl-CoA mutase; prpB, 2-methylcitrate lyase; prpC, 2-methylcitrate synthase; prpD, 2-methylcitrate dehydratase; prpE, propionyl-CoA synthetase; mmsA, methylmalonate semialdehyde dehydrogenase; hibA, acyl-CoA dehydrogenase; hibB, enoyl-CoA hydratase; mmsB, 3-hydroxyisobutyrate dehydrogenase; aceA, isocitrate dehydrogenase; aceB, malate synthase.

Tables

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  • TABLE 1

    PCC activity in extracts of photoheterotrophically grown cells

    StrainPCC activitya (nmol min−1 mg−1) with:
    SuccinateAcetatePropionate-HCO3−
    Wild type10 ± 176 ± 17207 ± 8
    RsΔpccRMC1228 ± 635 ± 2NGb
    RsΔpccRMC12(pMC66)1860191
    • ↵a Errors represent the standard deviations from at least three biological replicates.

    • ↵b NG, no growth.

  • TABLE 2

    LacZ activities in cell extracts of photoheterotrophically grown cells carrying derivatives of the pccB-lacZ translational fusion plasmid pMC85

    Strain and plasmidLacZ activitya (nmol/min/mg) with:
    SuccinateAcetatePropionate-HCO3−
    R. sphaeroides 2.4.1<5<5<5
        pMC85340 ± 304,350 ± 1,000NGb
        pMC85Δ110 ± 040 ± 090 ± 10
        pMC85Δ220 ± 030 ± 1030 ± 20
        pMC85Δ1230 ± 1060 ± 2060 ± 20
    RsΔpccRMC12<5<5NG
        pMC8520 ± 1050 ± 20NG
        pMC85Δ140 ± 2050 ± 10NG
        pMC85Δ220 ± 1040 ± 10NG
        pMC85Δ1230 ± 1060 ± 10NG
    • ↵a Errors represent the range for at least two biological replicates.

    • ↵b NG, no growth.

Additional Files

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  • Supplemental material

    • Supplemental file 1 -

      Table S1 (Primers), caption to Table S2, and Fig. S1 (CoA assimilation pathways), S2 (Sequencing reaction standards and the primer extension), S3 (Alignment of ScfR proteins), and S4 (Topography of the scaled neighbor-joining tree for 327 proteins from the ScfR family)

      PDF, 409K

    • Supplemental file 2 -

      Table S2 (Amino acid sequences of proteins and upstream regions)

      XLSX, 85K

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Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides
Michael S. Carter, Birgit E. Alber
Journal of Bacteriology Sep 2015, 197 (19) 3048-3056; DOI: 10.1128/JB.00402-15

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Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides
Michael S. Carter, Birgit E. Alber
Journal of Bacteriology Sep 2015, 197 (19) 3048-3056; DOI: 10.1128/JB.00402-15
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