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GENETICS AND MOLECULAR BIOLOGY

Regulation of Expression of Scaffoldin-Related Genes in Clostridium thermocellum

Tali W. Dror, Adi Rolider, Edward A. Bayer, Raphael Lamed, Yuval Shoham
Tali W. Dror
1Department of Food Engineering and Biotechnology, Technion—Israel Institute of Technology, Haifa
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Adi Rolider
1Department of Food Engineering and Biotechnology, Technion—Israel Institute of Technology, Haifa
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Edward A. Bayer
2Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot
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Raphael Lamed
3Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel
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Yuval Shoham
1Department of Food Engineering and Biotechnology, Technion—Israel Institute of Technology, Haifa
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  • For correspondence: yshoham@tx.technion.ac.il
DOI: 10.1128/JB.185.17.5109-5116.2003
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  • FIG. 1.
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    FIG. 1.

    Organization of C. thermocellum scaffoldin-related genes. cipA and the three genes olpB, orf2, and olpA are located in tandem within a gene cluster. sdbA is located on a distinct region of the chromosome. The proteins OlpB, Orf2p, and SdbA serve as anchoring proteins that connect the CipA scaffoldin to the cell surface. In contrast, OlpA selectively binds individual cellulases to the cell surface. The numbers above the gene in the scheme refer to its size (in base pairs).

  • FIG. 2.
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    FIG. 2.

    Transcript levels of the cipA gene and the genes coding for the cipA-specific anchoring proteins, olpB, orf2, and sdbA. RPAs were performed with the relevant 32P-labeled antisense probe as described earlier (11), using RNA from exponential (A) or stationary (B) phase cultures, grown on either cellobiose or crystalline cellulose. Different amounts (in micrograms) of RNA hybridized with the probes are indicated at the top of each lane. Autoradiographs of the respective RPA products were visualized using a phosphorimager system. Negative control lanes (NC) contained RNA from yeast. Lane M corresponds to markers (in base pairs). The full-length probe is shown in lane P. Values at right indicate the sizes of the undigested full-length antisense probes and the estimated sizes of the protected products in bases (b).

  • FIG. 3.
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    FIG. 3.

    mRNA expression of cipA and scaffoldin-related genes as determined by Northern blot analysis. Northern blot analysis was performed using total RNA isolated from cultures grown on either cellobiose (lanes 1 and 2) or crystalline cellulose (lanes 3 and 4), during exponential (lanes 1 and 3) and stationary (lanes 2 and 4) phases of growth. The isolated RNA (15 μg) was loaded and separated on agarose gels and then transferred to nitrocellulose membranes. The blot was hybridized subsequently with cipA, olpB, sdbA, and orf2 probes. The sizes of the16S and 23S rRNA and the estimated sizes of the transcripts are indicated in bases (b). Arrows denote larger-than-expected sized transcripts labeled by the designated probe (see text for details).

  • FIG. 4.
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    FIG. 4.

    Transcript levels of cipA and scaffoldin-related genes as a function of growth rate. The amounts of the respective mRNA were determined by RPA and converted to the number of transcripts per cell based on the average measured amount of total RNA in a single cell (0.18 pg/cell). Values represent the average of several (at least 3) measurements at an accuracy of ± 25%. Continuous cultures were operated under carbon (cellobiose) limitation at dilution rates between 0.04 ≤ μ ≤ 0.21; batch cultures were grown to exponential phase on either cellulose or cellobiose at rates of μ = 0.23 and 0.35 h−1, respectively.

  • FIG. 5.
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    FIG. 5.

    Mapping of the 5′ terminus of cipA by primer extension analysis. (A) 32P-labeled oligonucleotide was hybridized to mRNA obtained from C. thermocellum grown under the following conditions: continuous culture limited on cellobiose, diluted to a rate of 0.1 h−1 (lane 1); exponential-phase culture grown on Avicel (lane 2); and exponential-phase culture grown on cellobiose (lane 3). Dideoxynucleotide sequence reactions were performed using the same primers employed in the reverse transcriptase reactions. The positions of the transcriptional start points are indicated by asterisks on the inferred non-template-strand sequences. The products of primer extension are indicated by P1 and P2. (B) Sequence data for the regulatory region of cipA. The respective transcriptional start points (P1 and P2) are indicated. The consensus B. subtilis σA and σL promoter sequences are framed at the homologous sites of the C. thermocellum sequence. The proposed Shine-Dalgarno (SD) site and the initiating ATG codon are indicated.

Tables

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  • TABLE 1.

    Transcript levels of the scaffoldin gene, cipA, and the genes encoding for anchoring proteins olpB, orf2p, and sdbA in C. thermocellum cultures.

    GeneNo. of transcripts/cella
    CellobioseCrystalline cellulose
    ExponentialLate exponentialExponentialLate exponential
    cipA 98237
    olpB 24113
    orf2p 13141911
    sdbA 3446
    • ↵ a Values represent the average ± 15% standard error of at least three determinations of the mRNA level.

  • TABLE 2.

    Transcript levels of designated genes during continuous culture under nitrogen limitation

    GeneNo. of transcripts per cella at dilution rate (h−1)
    0.140.110.07
    cipA 81428
    olpB 91731
    orf2p 101425
    sdbA 3.93.55
    • ↵ a Values represent the average ± 15% standard error of at least three determinations of the mRNA level.

  • TABLE 3.

    Proposed promoter sequences in the regions upstream of selected cellulosome-related genes

    GeneProposed promoter sequencea
    cipA TAA-CACT—N16—TTGCTTGGTTTGT
    olpB TAAGCACT—N15—GTGATTACGACGA
    orf2p TAA CATA—N14—TTGCTGTTTTAAAGA
    celS TAA-CACA—N15—CAGCTTAGTTTTATG
    ConsensusTAA-CACW—Nn—NTGCTT
    Consensus σLTGG-CACN—N5—TTGCAb
    • ↵ a The −35 and −10 promoter sequences are shown to the left and right of extended intervening segments (Nn). Transcriptional start sites of cipA and celS (11) are designated in boldface type.

    • ↵ b Sequence from reference 46.

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Regulation of Expression of Scaffoldin-Related Genes in Clostridium thermocellum
Tali W. Dror, Adi Rolider, Edward A. Bayer, Raphael Lamed, Yuval Shoham
Journal of Bacteriology Aug 2003, 185 (17) 5109-5116; DOI: 10.1128/JB.185.17.5109-5116.2003

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Regulation of Expression of Scaffoldin-Related Genes in Clostridium thermocellum
Tali W. Dror, Adi Rolider, Edward A. Bayer, Raphael Lamed, Yuval Shoham
Journal of Bacteriology Aug 2003, 185 (17) 5109-5116; DOI: 10.1128/JB.185.17.5109-5116.2003
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KEYWORDS

Bacterial Outer Membrane Proteins
Bacterial Proteins
Capsid Proteins
Clostridium
Gene Expression Regulation, Bacterial
Glycoproteins
membrane proteins

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