A novel nucleoid protein of Escherichia coli induced under anaerobiotic growth conditions

Abstract

A systematic search was performed for DNA-binding sequences of YgiP, an uncharacterized transcription factor of Escherichia coli, by using the Genomic SELEX. A total of 688 YgiP-binding loci were identified after genome-wide profiling of SELEX fragments with a high-density microarray (SELEX-chip). Gel shift and DNase-I footprinting assays indicated that YgiP binds to multiple sites along DNA probes with a consensus GTTNATT sequence. Atomic force microscope observation indicated that at low concentrations, YgiP associates at various sites on DNA probes, but at high concentrations, YgiP covers the entire DNA surface supposedly through protein-protein contact. The intracellular concentration of YgiP is very low in growing E. coli cells under aerobic conditions, but increases more than 100-fold to the level as high as the major nucleoid proteins under anaerobic conditions. An E. coli mutant lacking ygiP showed retarded growth under anaerobic conditions. High abundance and large number of binding sites together indicate that YgiP is a nucleoid-associated protein with both architectural and regulatory roles as the nucleoid proteins Fis and IHF. We then propose that YgiP is a novel nucleoid protein of E. coli under anaerobiosis and propose to rename it Dan (DNA-binding protein under anaerobic conditions).

Figure 1.

Distribution of Dan-binding sequences on the E. coli genome. Mixtures of Dan-binding DNA fragments isolated by the genomic SELEX were subjected to the DNA microarray analysis. Binding signals (y-axis) are plotted against the location along the E. coli genome (x-axis). The genes showing high-level peaks above the cutoff level of 3.5 are indicated. The genes with asterisk represent those identified by SELEX-clos (see Supplementary Table 1).

Figure 2.

Gel mobility shift assay. Fluorescent-labeled DNA probes, each containing a SELEX segment from the dan-ttdA (A), maoC-paaA (B), yfaZ-yfaO (C), intZ-yffL (D), ygaD-[mltB]-srlA (E) and perR-insN (F) regions, or an unrelated reference fragment from yjbR-[uvrA]-ssb (G) and ykgI-ykgC spacer (H) were incubated at 37°C for 30 min with the indicated amounts (lane 1, 0; lane 2, 5 pmol; lane 3, 10 pmol; lane 4, 20 pmol; and lane 5, 30 pmol) of Dan. The reaction mixtures were directly subjected to PAGE.

Figure 3.

Identification of the Dan box sequence. (A) Fluorescent-labeled SELEX segment (1.0 pmol) from the dan-ttdA spacer was incubated in the absence or presence of increasing concentrations of purified Dan (10, 20 and 40 pmol from left to right) and then subjected to DNase-I foot-printing assays. Lanes A, T, G and C represent the respective sequence ladders. (B) Four Dan-binding sites were identified on the dan-ttdA spacer sequence. (C) The Dan-box sequence GTTAAT was predicted after sequence comparison of four Dan-binding sites. Similar sequences were identified among a total of 688 Dan-binding fragments selected by Genomic SELEX. Using this Dan-box sequence, a total of about 1860 sites were predicted to be present in the entire E. coli genome. (D) Logo representation of Dan-binding site derived from sequences in in silico analysis. Logos were generated using weblogo (http://weblogo.berkeley.edu/).

Figure 4.

AFM images of Dan–DNA complexes. (A–C) Dan-pGRdan DNA complexes were formed at input Dan/DNA weight ratios of 1/40 (A), 1/4 (B) and 1/1 (C), respectively. AFM images were taken by the standard procedure as described in ‘Materials and Methods’ section. Black arrowheads show naked DNA region, while white arrowheads highlight DNA-bound Dan molecules. All images represent 500 × 500 nm² area and scale bars indicate 100 nm. (D) Width of DNA-bound Dan dots was measured for a number of Dan dots observed in each AFM image. (E) DNA contour length was measured for a number of Dan–DNA complexes in each AFM image.

Figure 5.

Contour length of Dan–DNA complexes. (A) Dan–pGRdan DNA complexes were formed at various input Dan/DNA ratios (1/40, 1/20, 1/10, 1/4, 1/2, 1/1 and 1/0.25). Contour length of AFM images was measured for a number of complexes from each AFM image. The Dan–pGRdan complexes were classified into three groups based on the length: group-I, shorter than 960 nm; group-II, 960–2080 nm; and group-III, 2080–3200 nm. Data represent the mean value obtained from more than 100 samples (N > 100). (B) The relative amounts of three groups of Dan–DNA complex were plotted against the input Dan/DNA ratios.

Figure 6.

Intracellular concentration of the Dan protein. (A1) Wild-type E. coli KP7600 was grown in both LB and M9–0.4% glucose media at 37°C for various times under aerobic conditions. Cell lysates were prepared at various growth phases (lanes 1 and 7, log-phase at 0.3 OD₆₀₀; lanes 2 and 8, log-phase at 0.6 OD₆₀₀; lanes 3 and 9, late log-phase, 1.0 OD₆₀₀; lanes 4 and 10, 24-h stationary-phase; lane 5 and 11, 48-h stationary-phase; lanes 6 and 12, 72-h stationary-phase) and subjected to quantitative western blots analysis against anti-Dan and anti-RpoA antibodies. (A2) The intensity of immuno-blot bands was measured with a LAS-1000 Plus Lumino-Image analyzer and IMAGE GAUGE (Fuji Film). (B1) Wild-type E. coli KP7600 was grown in both LB and M9–0.4% glucose media at 37°C for various times under hypoxic culture conditions. Cell lysates were prepared at various growth phases (lanes 1 and 7, log-phase at 0.3 OD₆₀₀; lanes 2 and 8, log-phase at 0.6 OD₆₀₀; lanes 3 and 9, late log-phase, 1.0 OD₆₀₀; lanes 4 and 10, 24-h stationary-phase; lane 5 and 11, 48-h stationary-phase; lanes 6 and 12, 72-h stationary-phase) and subjected to western blots analysis against anti-Dan and anti-RpoA antibodies. (B2) The intensity of immuno-blot bands was measured with a LAS-1000 Plus Lumino-Image analyzer and IMAGE GAUGE (Fuji Film).

Figure 7.

Intracellular localization of the Dan protein. Wild-type E. coli KP7600 and its dan mutant JD24074 were grown in M9–0.4% glucose media for 24 h at 37°C under hypoxic conditions, and subjected to the indirect immuno-fluorescent microscopy according to the standard procedure described in ‘Materials and Methods’ section. (A) Wild-type strain KP7600 (W1–W4) and the dan mutant JD24074 (M1–M4) were grown under aerobic (A) or hypoxic (B) conditions. W1 and M1 represent; W2 and M2, Indirect immuno-blot against anti-Dan; W3 and M3, DAPI-staining; W4 and M4, merged images of DAPI and immuno-stained patterns. (C, D) The area shown by dotted square is expanded. Anti-Dan antibody was raised in rabbits using the purified Dan as an antigen. Cys3-labeled anti-rabbit IgG antibody was used as the secondary antibody. White bars indicate 5 μm.

Figure 8.

Model of Dan-induced DNA compaction. (A) On the bases of AFM image analysis of Dan-pGRdan DNA complexes formed at increasing concentrations of Dan (see Figure 1), we propose a model of the conformational change of plasmid DNA. At low Dan concentrations, Dan binds various positions on pGRdan in non-specific manner (Form I). Each Dan may play a role of seeding for further assembly of additional Dan molecules. At high Dan concentration, the increased number of Dan molecules bound to DNA strand, leading to extension of Dan oligomers along the DNA strand and ultimately covering the entire DNA surface (Form 2). Typical AFM images of form-I and form-II Dan-DNA complexes are shown on left (500 × 500 nm² area; scale bars, 100 nm). DNA strand is represented by a red line while Dan is represented by an orange rod. (B) A model of local conformation of DNA-bound Dan dots. Shortening of the contour length of pGRdan DNA might be due to local folding of DNA at the site of Dan binding (for details see text).