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1.
Figure 7.

Figure 7. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

Effect of overproduction of GnfL/GnfM on the expression of gnfK and gdhA. Total RNA was prepared from strains harboring the vector only, the GnfL/GnfM expression vector or the GnfM expression vector grown in the presence of . The expression of gnfK and gdhA was analyzed by primer extension assays.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
2.
Figure 9.

Figure 9. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

Growth under nitrogen-fixation condition. The wild-type (circles) and gnfK (triangles) or gnfR (squares) mutants were grown in the presence or absence of . Growth was monitored by measuring the optical density at 600 nm (OD600). Data are a representative of three replicate cultures.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
3.
Figure 6.

Figure 6. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

The two-component GnfL/GnfM system. (A) Purification of GnfLK and GnfM. The sizes of molecular mass standards are shown in kilodaltons. (B) In vitro phosphorylation assay. GnfLK and GnfM were tested for phosphotransfer activity in vitro. GnfLK was incubated at room temperature for 5 min for the autophosphorylation reaction. GnfM was then added and further incubated at room temperature for 5 min for the phosphotransfer reaction. The sizes of molecular mass standards are shown in kilodaltons.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
4.
Figure 12.

Figure 12. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

RNA binding of GnfR activated by GnfK. RNA-binding activity of GnfR was tested with RNA fragments containing the putative GnfR-binding site. (A) The wild-type RNA sequence. (B) A mutated RNA sequence. The RNA sequences of the putative GnfR-binding site are shown below the images of the gels. Mutated nucleotides are indicated with bold letters (see also Figure 10A). Lane 1, no protein; lane 2, GnfK; lane3, GnfR; lane 4, GnfR and GnfK.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
5.
Figure 2.

Figure 2. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

Identification of the master regulator for the nitrogen-fixation genes by DNA-binding assay. DNA fragments (40 fmol) containing upstream promoter regions of gnfK, nifH, glnB and gdhA were tested for DNA-binding activity of the EBPs (0.5 pmol) conserved in Geobacter species (Supplementary Table S3). The upstream promoter region of the GSU3409 gene was used as a control.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
6.
Figure 13.

Figure 13. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

Model for the regulation of gene expression during nitrogen fixation in G. sulfurreducens. Histidine kinases are presented in red. Response regulators are presented in blue. Phosphorylation is indicated in purple arrows. Transcription activation and repression are indicated in blue arrows and a pink line, respectively. Transcription antitermination is indicated by a green arrow. RNAP represents RNA polymerase.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
7.
Figure 8.

Figure 8. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

The two-component GnfK/GnfR system. (A) Purification of GnfK and GnfR. The sizes of molecular mass standards are shown in kilodaltons. (B) In vitro phosphorylation assay. The histidine kinase GnfK and the response regulator GnfR were tested for phosphotransfer activity in vitro. GnfK was incubated at a room temperature for 5 min for the autophosphorylation reaction. GnfR was then added and further incubated at a room temperature for 5 min for the phosphotransfer reaction. The sizes of molecular mass standards are shown in kilodaltons.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
8.
Figure 5.

Figure 5. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

Effect of the GnfM-binding sites on the expression of gnfK and gdhA. The promoter activity of gnfK (A) and gdhA (B) was analyzed by lacZ fusion assays. The activity is shown as a percentage of the activity of the wild-type promoter in the absence of for gnfK and in the presence of for gdhA. The location of GnfM-binding sites (BS) is indicated in Figure 4A and B. The binding sites were deleted (Δ) or mutated (M) in the assays. Diagrams of promoter constructs are shown and changes in the binding sites (M_BS3_BS4) are presented.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
9.
Figure 3.

Figure 3. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

GnfM-binding site. Footprint assays were carried out with GnfM and the upstream region of gnfK (A) or gdhA (B). DNA-binding reactions were conducted with GnfM (lane 1, 0 pmol; lane 2, 0.7 pmol; lane 3, 1.3 pmol; lane 4, 2.5 pmol for gnfK, lane 1, 0 pmol; lane 2, 0.3 pmol; lane 3, 0.7 pmol; lane 4, 1.3 pmol; lane 5, 2.5 pmol for gdhA) and probes (0.2 pmol). G, A, T and C represent sequence ladders. The region protected from or modified by DNase I in the presence of GnfM is indicated by a vertical bar.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
10.
Figure 11.

Figure 11. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

Transcription antitermination. (A) Diagrams of two transcripts of nifH mRNA. The locations of regions where primers (R1 and R2) used in the primer extension assay (B) hybridize are presented by arrows. (B) Expression of nifH and glnB. Total RNA was prepared from the wild-type and gnfK or gnfR mutants after they were grown to the early log phase in the media containing , harvested, resuspended and grown in the -free media. Equal amounts of total RNA for each strain were used to examine the expression of nifH and glnB by primer extension assays.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
11.
Figure 4.

Figure 4. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

Promoter regions. (A) gnfK. (B) gdhA. Regions modified in the presence of GnfM in the footprint assays (Figure 3) are highlighted in gray. Similar sequences found in the GnfM-binding sites are indicated by bold letters. Highly conserved dinucleotides GG and GC in RpoN-dependent −24/−12 promoter elements are underlined. Transcription initiation site is indicated by +1. Translation initiation codon is indicated by Met. (C) Alignment of similar sequences found in the GnfM-binding sites. (D) The consensus sequence of the GnfM-binding sites. The consensus sequence was obtained from the alignment of the putative GnfM-binding sites in the nitrogen-fixation genes (Supplementary Table S4) and is presented as a logo.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
12.
Figure 1.

Figure 1. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

RpoN-dependent promoter in nitrogen-fixation genes. (A) Expression of nitrogen-fixation genes and identification of their transcription initiation site. The expression of gnfK, gnfR, nifH, glnB and gdhA was examined by primer extension assays. Total RNA was prepared from cells grown in the presence (+) or absence (−) of . G, A, T and C represent sequence ladders generated by the same primer used in the primer extension assays. (B) RpoN-dependent −24/−12 promoter elements of the nitrogen-fixation genes. The −24/−12 promoter elements for nifH, glnB, gnfK, gnfR and gdhA of G. sulfurreducens were assigned from the transcription initiation sites (+1) determined by the primer extension assays (A). (C) The consensus sequence of the −24/−12 promoter elements. The consensus sequence was obtained from the alignment shown in Supplementary Table S2 and is presented as a logo.

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.
13.
Figure 10.

Figure 10. From: Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

Premature transcription termination in the nitrogen-fixation genes. (A) Alignment of transcription termination-like signals in the nitrogen-fixation genes. It is likely that the nifEN operon contains nifEN and nifX in this order, the Gsul_0938 homologue operon contains a gene encoding a hypothetical protein, glnK and amtB in this order, and the Gsul_2799 homologue operon contains genes encoding a putative radical SAM domain protein and a putative acetyltransferase. The inverted-repeat sequences are underlined. Highly conserved nucleotides are indicated in bold letters. (B) Promoter region of nifH. Predicted GnfM-binding sites (Supplementary Table S4) are highlighted in gray. Highly conserved dinucleotides GG and GC in RpoN-dependent −24/−12 promoter elements are underlined. Transcription initiation site is indicated by +1. Transcription termination-like signals are indicated by arrows. Translation initiation codon is indicated by Met. (C) Putative RNA secondary structure of nifH. The putative stem-loop structure was predicted on the basis of base-pairing. A predicted binding site for GnfR is indicated in bold letters. (D) lacZ fusion assay. The assays were conducted with artificial promoters. Diagrams of promoter constructs are shown (see Supplementary Figure S5A for sequences). RpoD, the RpoD-dependent like promoter; RpoD-TTS (WT), the RpoD-dependent like promoter with the wild-type transcription termination signals from nifH; RpoD-TTS (-up), the RpoD-dependent like promoter with the transcription termination signals lacking the upstream half of the stem structure; RpoD-TTS (-down), the RpoD-dependent like promoter with the transcription termination signals lacking the downstream half of the stem structure; RpoD-TTS (mut), the RpoD-dependent like promoter with the transcription termination signals mutated in the stem structure (Supplementary Figure S5A). Mutations in RpoD-TTS (mut) were made by changing GGGGCGCCTT to ACATACAACA. β-Galactosidase activity was examined with the X-gal plate (Supplementary Figure S5B).

Toshiyuki Ueki, et al. Nucleic Acids Res. 2010 November;38(21):7485-7499.

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