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. 2019 Dec 31;9(1):20415.
doi: 10.1038/s41598-019-56886-x.

Regulatory Role of PlaR (YiaJ) for Plant Utilization in Escherichia coli K-12

Tomohiro Shimada  1   2 , Yui Yokoyama  3 , Takumi Anzai  4 , Kaneyoshi Yamamoto  3 , Akira Ishihama  5   6
Affiliations

Regulatory Role of PlaR (YiaJ) for Plant Utilization in Escherichia coli K-12

Tomohiro Shimada et al. Sci Rep. .

Erratum in

Abstract

Outside a warm-blooded animal host, the enterobacterium Escherichia coli K-12 is also able to grow and survive in stressful nature. The major organic substance in nature is plant, but the genetic system of E. coli how to utilize plant-derived materials as nutrients is poorly understood. Here we describe the set of regulatory targets for uncharacterized IclR-family transcription factor YiaJ on the E. coli genome, using gSELEX screening system. Among a total of 18 high-affinity binding targets of YiaJ, the major regulatory target was identified to be the yiaLMNOPQRS operon for utilization of ascorbate from fruits and galacturonate from plant pectin. The targets of YiaJ also include the genes involved in the utilization for other plant-derived materials as nutrients such as fructose, sorbitol, glycerol and fructoselysine. Detailed in vitro and in vivo analyses suggest that L-ascorbate and α-D-galacturonate are the effector ligands for regulation of YiaJ function. These findings altogether indicate that YiaJ plays a major regulatory role in expression of a set of the genes for the utilization of plant-derived materials as nutrients for survival. PlaR was also suggested to play protecting roles of E. coli under stressful environments in nature, including the formation of biofilm. We then propose renaming YiaJ to PlaR (regulator of plant utilization). The natural hosts of enterobacterium Escherichia coli are warm-blooded animals, but even outside hosts, E. coli can survive even under stressful environments. On earth, the most common organic materials to be used as nutrients by E. coli are plant-derived components, but up to the present time, the genetic system of E. coli for plant utilization is poorly understand. In the course of gSELEX screening of the regulatory targets for hitherto uncharacterized TFs, we identified in this study the involvement of the IclR-family YiaJ in the regulation of about 20 genes or operons, of which the majority are related to the catabolism of plant-derived materials such as ascorbate, galacturonate, sorbitol, fructose and fructoselysine. Therefore, we propose to rename YiaJ to PlaR (regulator of plant utilization).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Mapping of PlaR-binding sites along the E. coli K-12 genome. (a) gSELEX screening for 5-cycles; (b) gSELEX screening for 6-cycles. gSELEX screening of PlaR-binding sequences was performed after which, a collection of DNA fragments was subjected to chip analysis using the tilling array of the E. coli K-12 genome. The y-axis represents the relative number of PlaR-bound DNA fragments, and the x-axis represents the position on the E. coli genome. The locations of PlaR-binding sites on the intergenic region are shown. The detailed PlaR-binding sites are listed in Fig. 2.
Figure 2
Figure 2
PlaR-binding sites on the E. coli genome were identified by using gSELEX-chip (see Fig. 1 for the gSELEX pattern). Possible regulation targets of PlaR are shown in bold, of which identified SELEX 5-cycles are shown under pale and dark green background, and SELEX 6-cycles are shown under dark green background.
Figure 3
Figure 3
Confirmation of PlaR-binding in vitro to the regulatory targets: Gel shift assay. Purified PlaR was mixed with 0.5 pM of each DNA probe corresponding to the PlaR-binding regions shown in Fig. 2. PlaR (μM) was added: lane 1, 0; lane 2, 1; lane 3, 2.5; lane 4, 5. Filled triangles indicate the PlaR-DNA probe complex, and arrows indicates free probe.
Figure 4
Figure 4
Consensus sequence of PlaR-box. Sequences of the probes with high level of PlaR-binding activity were analyzed using DMINDA 2.0 program (http://bmbl.sdstate.edu/DMINDA2/) and WEBLOGO (http://weblogo.berkeley.edu/logo.cgi) was used for matrix construction.
Figure 5
Figure 5
Influence of PlaR on transcription of the regulatory target genes. E. coli wild-type BW25113 (lane 1) and its plaR-defective mutant (lane 2) and kdgR-defective mutant (lane 3) were grown in M9-casamino acids (0.5%) medium at 37 °C with shaking under aerobic conditions. In the middle of exponential phase, total RNA was extracted from each culture and subjected to Northern blot analysis. DIG-labelled hybridization probes are shown on the left side of each panel. The amounts of total RNA analyzed were determined by measuring the intensity of ribosomal RNAs.
Figure 6
Figure 6
Repoter assay of the yiaK promoter. Reporter assay of the yiaK promoter was carried out using the lacZ reporter encoding β-galactosidase. Single copy lacZ gene reporter strains containing yiaK-lacZ was constructed both in wild-type strain (a) and plaR deleted strain (b). The strain was grown in M9 medium supplemented with 0.5% casamino acids. β-galactosidase activity was measured 1 h after addition of each effector in the middle of the exponential phase. 0.2% of α-D-galacturonate or 30 mM of L-ascorbate was supplemented, respectively. The data represent mean values from three separate experiments.
Figure 7
Figure 7
Influence of PlaR on cell growth in the presence of difference carbon sources. E. coli K-12 wild-type BW25113, plaR deleted mutant (a), and wild-type with IPTG-inducible PlaR-expression plasmid (b1 to d1) or vector plasmid pCA24N (b2 to d2) were grown in M9 medium containing 0.2% of galacturonate (b), sorbitol (c) or fructose (d). Various IPTG concentrations were added to evaluate the influence of PlaR expression level (b1 to d2). Cell growth was monitored by following the culture turbidity at 600 nm. The average of triplicate experiments is shown.
Figure 8
Figure 8
Search for inducer candidates for derepression of the yiaK promoter by PlaR. 2.5 uM of Purified PlaR was mixed with 0.5 pM of a DNA probe corresponding to the promoter sequence of yiaK gene. The yiaK promoter formed stable complexes with PlaR as shown in Fig. 3. Possible inducers affecting the yiaK promoter-PlaR complex formation, a variety of carbon sources were examined using the gel shift assay system. (Panel a) Eight species of carbon metabolite were tested at 10 mM concentrations. (Panel b) influence of two effective metabolites, ascorbate and D-galacturonate, was examined in details by increasing concentrations. (b) Ascorbate or D-galacturonate (mM) was added: lane 3, 1; lane 4, 2.5; lane 5, 5; lane 6, 10; lane 7, 25, respectively. Filled arrow indicates PlaR-yiaK promoter probe complex and open arrow indicates free probe.
Figure 9
Figure 9
Participation of PlaR-target genes in the catabolism of D-galacturonate and L-ascorbate. The regulatory target genes were identified by gSELEX screening. These PlaR-target genes are involved in the catabolism of L-galacturonate and L-ascorbate. L-Galacturonate is derived from plant cell-wall pectin after digestion by pectinases from plant pathogens and transported into E. coli while L-ascorbate from fruits is spontaneously oxidized and taken up by E. coli through the YiaMNO importer. The genes under the direct control of PlaR are shown with under red background.
Figure 10
Figure 10
Regulatory roles of hitherto uncharacterized Y-TFs in utilization of plant-derived nutrients. After gSELEX screening of regulatory targets of hitherto uncharacterized TFs, we have identified the involvement of some TFs in regulation of a group of genes that participate in utilization of plant-derived materials. Here we identified the regulation by PlaR (renamed YiaJ) of genes for utilization of L-ascorbate from plant fruits (a). PlaR was also indicated to regulate genes participating in the utilization of D-galacturonate derived from plant cell-wall lectin and sorbital from fruit (b). Previously we identified the involvement of CsqR (renamed YihW) in the catabolism of fulfolipid from plant chloroplast (Shimada et al.) and XynR (renamed YagI) in the catabolism of xylan from plant cell wall (Shimada et al.) (c).
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