Investigating the DNA-Binding Site for VirB, a Key Transcriptional Regulator of Shigella Virulence Genes, Using an In Vivo Binding Tool

The transcriptional anti-silencing and DNA-binding protein, VirB, is essential for the virulence of Shigella species and, yet, sequences required for VirB-DNA binding are poorly understood. While a 7-8 bp VirB-binding site has been proposed, it was derived from studies at a single VirB-dependent promoter, icsB. Our previous in vivo studies at a different VirB-dependent promoter, icsP, found that the proposed VirB-binding site was insufficient for regulation. Instead, the required site was found to be organized as a near-perfect inverted repeat separated by a single nucleotide spacer. Thus, the proposed 7-8 bp VirB-binding site needed to be re-evaluated. Here, we engineer and validate a molecular tool to capture protein-DNA binding interactions in vivo. Our data show that a sequence organized as a near-perfect inverted repeat is required for VirB-DNA binding interactions in vivo at both the icsB and icsP promoters. Furthermore, the previously proposed VirB-binding site and multiple sites found as a result of its description (i.e., sites located at the virB, virF, spa15, and virA promoters) are not sufficient for VirB to bind in vivo using this tool. The implications of these findings are discussed

A review of environmental contamination and health risk assessment of wastewater use for crop irrigation with a focus on low and high-income countries

Population densities and freshwater resources are not evenly distributed worldwide. This has forced farmers to use wastewater for the irrigation of food crops. This practice presents both positive and negative effects with respect to agricultural use, as well as in the context of environmental contamination and toxicology. Although wastewater is an important source of essential nutrients for plants, many environmental, sanitary, and health risks are also associated with the use of wastewater for crop irrigation due to the presence of toxic contaminants and microbes. This review highlights the harmful and beneficial impacts of wastewater irrigation on the physical, biological, and chemical properties of soil (pH, cations and anions, organic matter, microbial activity). We delineate the potentially toxic element (PTEs) build up in the soil and, as such, their transfer into plants and humans. The possible human health risks associated with the use of untreated wastewater for crop irrigation are also predicted and discussed. We compare the current condition of wastewater reuse in agriculture and the associated environmental and health issues between developing and developed countries. In addition, some integrated sustainable solutions and future perspectives are also proposed, keeping in view the regional and global context, as well as the grounded reality of wastewater use for crop production, sanitary and planning issues, remedial techniques, awareness among civil society, and the role of the government and the relevant stakeholders

The AraC/XylS protein MxiE and its co-regulator IpgC control a negative feedback loop in the transcriptional cascade that regulates type III secretion in Shigella flexneri

Members of the AraC Family of Transcriptional Regulators (AFTRs) control the expression of many genes important to cellular processes, including virulence. In Shigella species, the type III secretion system (T3SS), a key determinant for host cell invasion, is regulated by the three-tiered VirF/VirB/MxiE transcription cascade. Both VirF and MxiE belong to the AFTRs and are characterized as positive transcriptional regulators. Here, we identify a novel regulatory activity for MxiE and its co-regulator IpgC, which manifests as a negative feedback loop in the VirF/VirB/MxiE transcription cascade. Our findings show that MxiE and IpgC down-regulate the virB promoter and hence VirB protein production, thus, decreasing VirB-dependent promoter activity at ospD1, one of the nearly 50 VirB-dependent genes. At the virB promoter, regions required for negative MxiE- and IpgC-dependent regulation were mapped and found to be coincident with regions required for positive VirF-dependent regulation. In tandem, negative MxiE- and IpgC-dependent regulation of the virB promoter only occurred in the presence of VirF suggesting that MxiE and IpgC can function to counter VirF activation of the virB promoter. Lastly, MxiE and IpgC do not down-regulate another VirF-activated promoter, icsA, demonstrating that this negative feedback loop targets the virB promoter. Our study provides insight into a mechanism that may reprogram Shigella virulence gene expression following type III secretion and provides the impetus to examine if MxiE and IpgC homologs in other important bacterial pathogens such as Burkholderia pseudomallei and Salmonella enterica serovars Typhimurium and Typhi coordinate similar negative feedback loops

The AraC/XylS Protein MxiE and Its Coregulator IpgC Control a Negative Feedback Loop in the Transcriptional Cascade That Regulates Type III Secretion in Shigella flexneri

The large AraC family of transcriptional regulators (AFTRs) control virulence gene expression in many bacterial pathogens. In Shigella species, the AraC/XylS protein MxiE and its coregulator IpgC positively regulate the expression of type III secretion system genes within the three-tiered VirF/VirB/MxiE transcriptional cascade. ABSTRACT Members of the AraC family of transcriptional regulators (AFTRs) control the expression of many genes important to cellular processes, including virulence. In Shigella species, the type III secretion system (T3SS), a key determinant for host cell invasion, is regulated by the three-tiered VirF/VirB/MxiE transcriptional cascade. Both VirF and MxiE belong to the AFTRs and are characterized as positive transcriptional regulators. Here, we identify a novel regulatory activity for MxiE and its coregulator IpgC, which manifests as a negative feedback loop in the VirF/VirB/MxiE transcriptional cascade. Our findings show that MxiE and IpgC downregulate the virB promoter and, hence, VirB protein production, thus decreasing VirB-dependent promoter activity at ospD1 , one of the nearly 50 VirB-dependent genes. At the virB promoter, regions required for negative MxiE- and IpgC-dependent regulation were mapped and found to be coincident with regions required for positive VirF-dependent regulation. In tandem, negative MxiE- and IpgC-dependent regulation of the virB promoter only occurred in the presence of VirF, suggesting that MxiE and IpgC can function to counter VirF activation of the virB promoter. Lastly, MxiE and IpgC do not downregulate another VirF-activated promoter, icsA , demonstrating that this negative feedback loop targets the virB promoter. Our study provides insight into a mechanism that may reprogram Shigella virulence gene expression following type III secretion and provides the impetus to examine if MxiE and IpgC homologs in other important bacterial pathogens, such as Burkholderia pseudomallei and Salmonella enterica serovars Typhimurium and Typhi, coordinate similar negative feedback loops. IMPORTANCE The large AraC family of transcriptional regulators (AFTRs) control virulence gene expression in many bacterial pathogens. In Shigella species, the AraC/XylS protein MxiE and its coregulator IpgC positively regulate the expression of type III secretion system genes within the three-tiered VirF/VirB/MxiE transcriptional cascade. Our findings suggest a negative feedback loop in the VirF/VirB/MxiE cascade, in which MxiE and IpgC counter VirF-dependent activation of the virB promoter, thus making this the first characterization of negative MxiE- and IpgC-dependent regulation. Our study provides insight into a mechanism that likely reprograms Shigella virulence gene expression following type III secretion, which has implications for other important bacterial pathogens with functional homologs of MxiE and IpgC

Mg2+ facilitates leader peptide translation to induce riboswitch-mediated transcription termination

The 5′-UTR of the Salmonella Mg2+ transporter mgtA contains a magnesium sensitive riboswitch encompassing the ORF mgtL. MGTL translation is regulated by Mg2+-dependent opening of a ribosome-binding site and causes premature termination of mgtA transcription in cis