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. 2023 Jan 7;24(2):1184.
doi: 10.3390/ijms24021184.

Insight into the Global Negative Regulation of Iron Scavenger 7-HT Biosynthesis by the SigW/RsiW System in Pseudomonas donghuensis HYS

Shiyu Teng  1 , Tingting Wu  1 , Donghao Gao  1 , Siyi Wu  1 , Yaqian Xiao  1 , Yan Long  1 , Zhixiong Xie  1
Affiliations

Insight into the Global Negative Regulation of Iron Scavenger 7-HT Biosynthesis by the SigW/RsiW System in Pseudomonas donghuensis HYS

Shiyu Teng et al. Int J Mol Sci. .

Abstract

7-Hydroxytropolone (7-HT) is a unique iron scavenger synthesized by Pseudomonas donghuensis HYS that has various biological activities in addition to functioning as a siderophore. P. donghuensis HYS is more pathogenic than P. aeruginosa toward Caenorhabditis elegans, an observation that is closely linked to the biosynthesis of 7-HT. The nonfluorescent siderophore (nfs) gene cluster is responsible for the orderly biosynthesis of 7-HT and represents a competitive advantage that contributes to the increased survival of P. donghuensis HYS; however, the regulatory mechanisms of 7-HT biosynthesis remain unclear. This study is the first to propose that the ECF σ factor has a regulatory effect on 7-HT biosynthesis. In total, 20 ECF σ factors were identified through genome-wide scanning, and their responses to extracellular ferrous ions were characterized. We found that SigW was both significantly upregulated under high-iron conditions and repressed by an adjacent anti-σ factor. RNA-Seq results suggest that the SigW/RsiW system is involved in iron metabolism and 7-HT biosynthesis. Combined with the siderophore phenotype, we also found that SigW could inhibit siderophore synthesis, and this inhibition can be relieved by RsiW. EMSA assays proved that SigW, when highly expressed, can directly bind to the promoter region of five operons of the nfs cluster to inhibit the transcription of the corresponding genes and consequently suppress 7-HT biosynthesis. In addition, SigW not only directly negatively regulates structural genes related to 7-HT synthesis but also inhibits the transcription of regulatory proteins, including of the Gac/Rsm cascade system. Taken together, our results highlight that the biosynthesis of 7-HT is negatively regulated by SigW and that the SigW/RsiW system is involved in mechanisms for the regulation of iron homeostasis in P. donghuensis HYS. As a result of this work, we identified a novel mechanism for the global negative regulation of 7-HT biosynthesis, complementing our understanding of the function of ECF σ factors in Pseudomonas.

Keywords: 7-hydroxytropolone; Pseudomonas donghuensis HYS; SigW; anti-σ factor; negative regulator; siderophore.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Screening of the ECF σ factors involved in iron metabolism in P. donghuensis HYS. RNA was isolated from the indicated strains grown to the exponential phase at 30 °C in liquid MKB culture with or without 30 μM FeSO4·7H2O supplementation. The error bars indicate the mean ± SD of three independent experiments. Statistical significance was calculated using one-way ANOVA Dunnett’s multiple comparison test, ** p < 0.001; *** p < 0.0001. (A) The P. donghuensis strain HYS σ factors regulome. (B) Expression of 20 ECF σ factors encoded on the genome in different extracellular iron environments. (C) Genetic organization of σ16 and sigW. The locus tags of the corresponding genes in P. putida NBRC 14164 (accession number NC_021505) and P. fluorescens ATCC 13525 (accession number NZ_LT907842.1) are shown under the arrows.
Figure 2
Figure 2
RsiW functions as the anti-σ factor of SigW in P. donghuensis HYS. The domain architectures are illustrated, along with their structures. The region 2 and region 4 domains usually carried by ECF σ factors are shown. The blue lines represent DNA binding sites with the corresponding amino acid indicated below (A). The transmembrane helices (TMH) in RsiW were predicted by the transmembrane protein topology prediction tool TMHMM (B). (C) Expression of the anti-σ factor, rsiW in different extracellular iron environments. MKB simulates an iron-limited environment and MKB supplement 30 μM ferrous ions simulates an iron-rich environment. (D) The relative expression of 20 ECF σ factors in the wild-type strain of P. donghuensis HYS and ΔrsiW mutant under the iron-limited conditions. RNA was isolated from the indicated strains grown to the exponential phase at 30 °C in liquid MKB culture with or without 30 μM FeSO4·7H2O supplementation. The error bars indicate the mean ± SD of three independent experiments. Statistical significance was calculated using one-way ANOVA Dunnett’s multiple comparison test, *** p < 0.0001.
Figure 3
Figure 3
Transcriptional profiling with RNA sequencing identified SigW/RsiW regulated genes in P. donghuensis HYS. Wild-type strains lacking sigW or rsiW or carrying pBBR2-sigW (overexpressing sigW), grown in MKB medium were analyzed by RNA sequencing. (A) Stacking diagram representing the number of differential genes among the three groups. The black and grey bars represent down- and upregulated genes, respectively. In the three comparison groups, the total number of differential genes was 66, 585, and 1032, respectively. The graph was created based on the mean value of fold changes in triplicates. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (B) (ΔsigW vs. HYS) and (C) (HYS/pBBR2−sigW vs. HYS/pBBR2). The black and grey bars represent down- and upregulated genes, respectively, while the bars represent the number of genes related to that pathway. Selection of enrichment pathways with p values less than 0.05 is shown in the histogram. p-values < 0.05 indicate that the function was significantly enriched. (D) Heatmap demonstrating a selection of genes differentially expressed in the comparisons are indicated above each column. The diagram was based on the mean value of fold changes in triplicates.
Figure 4
Figure 4
Effects of sigW and rsiW on siderophores and biofilm formation in P. donghuensis HYS. (A) Absorption spectra of the filtered supernatants of 24 h MKB cultures from wild-type HYS and the derivative strains. 7-HT has characteristic absorption at 330 and 392 nm, and pyoverdine has characteristic absorption at 405 nm. (B) Pyoverdine production in 24 h MKB cultures of wild-type HYS, ΔsigW, and ΔrsiW mutants. (C) Siderophore production in wild-type HYS and the derivative strains in liquid MKB medium was determined as siderophore units (percent) by the CAS liquid assay. (D) Deletion of rsiW decreased biofilm formation. The biofilm formation of wild-type HYS, ΔrsiW mutant, and the complemented strain was detected using crystal violet staining (lower) and quantified b optical density measurement (upper). The error bars indicate the mean ± SD of three independent experiments. Statistical significance was calculated using one-way ANOVA Dunnett’s multiple comparison test, * p < 0.01; ** p < 0.001.
Figure 5
Figure 5
SigW binds directly to promoters on the nfs cluster for regulatory action. Localization map of promoters on the nfs cluster (A). The electrophoretic mobility shift assay shows that SigW binds to the promoter region of the wild-type (B) orf1, (C) orf2–5, (D) orf9–6, (E) orf10–11, and (F) orf12, respectively. Each reaction mixture contained 0.3 μM PCR products of the wild-type orf1–217 to –1, orf2–191 to –1, orf9–225 to –1, orf10–237 to –1, orf12–246 to –1, and PD2720–210 to –1. The protein concentrations are indicated above the lane. BSA and P-2720 were used as negative controls. Data are representative of three independent replicates.
Figure 6
Figure 6
Validation of the nfs cluster regulated by SigW at the transcriptional levels. The results show the relative expression levels of the orf1 and orf12 (A), orf2–orf5 (B), orf9–orf6 (C), orf10–orf11 (D) in HYS/pBBR2, HYS/pBBR2-sigW, ΔsigW mutant, and wild-type HYS. The transcriptional levels are shown as the relative expression of genes compared to the expression of the rpoB gene in various samples at the exponential phase, as measured by qRT-PCR. Error bars indicate the mean ± SD of three independent experiments. Statistical significance was calculated using one-way ANOVA Dunnett’s multiple comparison test, * p < 0.01; ** p < 0.001; *** p < 0.0001.
Figure 7
Figure 7
Genetic organization and characteristics of the sigW operon. (A) The 5′-RACE method was used to identify the TSS of the sigW operon using RNA sample, with the −10 and −35 motifs then deduced afterwards. The red triangle represents the start codon. The genes are drawn to scale. (B) MEME online prediction of the conserved motifs of SigW. The seqlogo plot shows how well the motif is conserved at each position; the higher the letter, the better the position is conserved. Different amino acids in the same position are scaled according to their frequency. The rules for construction logos are given B–C or G or T, Y–C or T, S–G or C, D–A or G or T, W–A or T, K–G or T. This graph is based on a motif sequence with an E-value less than or equal to 0.05. Further, MEME-Suite was used to predict motif information in the SigW sequence, as conserved motifs on transcription factors are usually involved in important biological processes. By submitting 20 amino acid sequences with >80% identity to SigW online, the output resulted in four highly conserved motifs with the second motif (34–83 bp) and fourth motif (120–169 bp) located in conserved regions 2 and 4, respectively, of the σ70 family in Pseudomonas (Figure 7B). In summary, the transcription structure information of this operon was clarified, and prediction of the −10, −35 regions, and conserved motifs is helpful for subsequent functional verification. In particular, the predicted conserved motifs clarified that this manipulator has a structure that is typical of the ECF σ factors.
Figure 8
Figure 8
The regulatory relationship between SigW and the Gac/Rsm cascade system. Expression of gacA/S, rsmA/E, and rsmY/Z in P. donghuensis HYS. RNA was isolated from the indicated strains grown to the exponential phase at 30 °C in liquid MKB culture. Error bars indicate the mean ± SD of three independent experiments. Statistical significance was calculated using one-way ANOVA Dunnett’s multiple comparison test, * p < 0.01; *** p < 0.0001.
Figure 9
Figure 9
Schematic overview of the regulatory network of SigW/RsiW in P. donghuensis HYS. The T-shaped lines represent the negative control, the arrows represent the positive control, the solid lines highlight the existence of an already demonstrated regulation, and the dotted lines indicate the connections that were not confirmed in this work. P1, P2, P9, P10, and P12 represent the promoters of the five operons in the nfs cluster, respectively. ORF6, ORF7, ORF8, and ORF9 encode the Pdc, PaaK, CaiA, and FadM family proteins, respectively, which catalyze the key reaction of 7-HT biosynthesis.
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