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Scanning iron response regulator binding sites using Dap-seq in the Brucella genome.

ABSTRACT: Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Previous studies suggested that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, in the present study, a Dap-seq approach was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes was identified using qRT-PCR. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), ftrA (BME_RS14525), cation-transporting P-type ATPase (zntA) (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. Furthermore, the iron utilization and growth assay experiments confirmed that rirA was involve in iron metabolism and growth of Brucella. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism, and the rirA was identified as a regulon of Irr and it also plays a role in iron metabolism of Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism.

SUBMITTER: Zhang H

PROVIDER: S-EPMC10374146 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Scanning iron response regulator binding sites using Dap-seq in the Brucella genome.

Zhang Huan H Sun Tianhao T Cao Xudong X Wang Yifan Y Ma Zhongchen Z Wang Yueli Y Yang Ningning N Xu Mingguo M Deng Xiaoyu X Li Honghuan H Wang Benben B Yi Jihai J Wang Zhen Z Zhang Qian Q Chen Chuangfu C

PLoS neglected tropical diseases 20230717 7

Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In additio ...[more]

PMID: 37459300

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Project description:Brucella spp. are gram-negative, facultative intracellular pathogens that cause brucellosis in humans and animals. Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Currently, bhuA (a heme transporter), bhuQ (heme utilization oxygenase Q), and RirA (iron-responsive regulator) have been identified as iron metabolism genes and are regulated by the Irr protein. These results suggest that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, a combination of RNA-seq and Dap-seq was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes were similar in both the RNA-seq and qRT-PCR results. Furthermore, the DNA bases C and G and amino acids asparagine, arginine, histidine, and serine were predicted as predominant docking sites for DNA and Irr using the HDOCK server. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), Iron transporter (BME_RS14525), cation-transporting P-type ATPase (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism. In addition, from the RNA-seq results, the mutation of irr resulted in the down/upregulation of many genes that are associated with the bacterial secretion system, lipopolysaccharides, and flagellar assembly, and these three components are the main virulence factors for Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism and its effects on virulence.

Project description:ChIP-Seq, which combines chromatin immunoprecipitation (ChIP) with ultra high-throughput massively parallel sequencing, is increasingly being used for mapping protein-DNA interactions in-vivo on a genome scale. Typically, short sequence reads from ChIP-Seq are mapped to a reference genome for further analysis. Although genomic regions enriched with mapped reads could be inferred as approximate binding regions, short read lengths (approximately 25-50 nt) pose challenges for determining the exact binding sites within these regions. Here, we present SISSRs (Site Identification from Short Sequence Reads), a novel algorithm for precise identification of binding sites from short reads generated from ChIP-Seq experiments. The sensitivity and specificity of SISSRs are demonstrated by applying it on ChIP-Seq data for three widely studied and well-characterized human transcription factors: CTCF (CCCTC-binding factor), NRSF (neuron-restrictive silencer factor) and STAT1 (signal transducer and activator of transcription protein 1). We identified 26 814, 5813 and 73 956 binding sites for CTCF, NRSF and STAT1 proteins, respectively, which is 32, 299 and 78% more than that inferred previously for the respective proteins. Motif analysis revealed that an overwhelming majority of the identified binding sites contained the previously established consensus binding sequence for the respective proteins, thus attesting for SISSRs' accuracy. SISSRs' sensitivity and precision facilitated further analyses of ChIP-Seq data revealing interesting insights, which we believe will serve as guidance for designing ChIP-Seq experiments to map in vivo protein-DNA interactions. We also show that tag densities at the binding sites are a good indicator of protein-DNA binding affinity, which could be used to distinguish and characterize strong and weak binding sites. Using tag density as an indicator of DNA-binding affinity, we have identified core residues within the NRSF and CTCF binding sites that are critical for a stronger DNA binding.

Dataset Information

Scanning iron response regulator binding sites using Dap-seq in the Brucella genome.

Publications

Scanning iron response regulator binding sites using Dap-seq in the Brucella genome.

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