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Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2., Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2., Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

In response to nutrient limitation in the environment, the global transcriptional regulator CodY modulates various pathways in low G+C Gram-positive bacteria. In Bacillus subtilis CodY triggers adaptation to starvation by secretion of proteases coupled to the expression of amino acid transporters. Furthermore, it is involved in modulating survival strategies like sporulation, motility, biofilm formation, and CodY is also known to affect virulence factor production in pathogenic bacteria. In this study, the role of CodY in Bacillus cereus ATCC 14579, the enterotoxin-producing type strain, is investigated. A marker-less deletion mutant of codY (?codY) was generated in B. cereus and the transcriptome changes were surveyed using DNA microarrays. Numerous genes involved in biofilm formation and amino acid transport and metabolism were upregulated and genes associated with motility and virulence were repressed upon deletion of codY. Moreover, we found that CodY is important for efficient production of toxins and for adapting from nutrient-rich to nutrient-limited growth conditions of B. cereus. In contrast, biofilm formation is highly induced in the ?codY mutant, suggesting that CodY represses biofilm formation. Together, these results indicate that CodY plays a crucial role in the growth and persistence of B. cereus in different environments such as soil, food, insect guts and the human body.

We characterized a new quorum-sensing regulator, PlcRa, which is present in various members of the B. cereus group and identified a signaling heptapeptide for PlcRa activity: PapRa7. We demonstrated that PlcRa is a 3D structural paralog of PlcR using sequence analysis and homology modeling. A comparison of the transcriptomes at the onset of stationary phase of a ¿plcRa mutant and the wild-type B. cereus ATCC 14579 strain showed that 68 genes were upregulated and 49 genes were downregulated in the ¿plcRa mutant strain (>3-fold change). Genes involved in the cysteine metabolism (putative CymR regulon) were downregulated in the ¿plcRa mutant strain. We focused on the gene with the largest difference in expression level between the two conditions, which encoded -AbrB2- a new regulator of the AbrB family. We demonstrated that purified PlcRa bound specifically to the abrB2 promoter in the presence of synthetic PapRa7, in an electrophoretic mobility shift assay. We further showed that the AbrB2 regulator controlled the expression of the yrrT operon involved in methionine to cysteine conversion. We found that the ¿plcRa mutant strain was more sensitive to hydrogen peroxide- and disulfide-induced stresses than the wild type. When cystine was added to the culture of the ¿plcRa mutant, challenged with hydrogen peroxide, growth inhibition was abolished. In conclusion, we identified a new RNPP transcriptional regulator in B. cereus that activated the oxidative stress response and cysteine metabolism in transition state cells.

Understanding the mechanisms by which transcriptional regulatory networks (TRNs) change through evolution is a fundamental problem.Here, we analyze this question using data from Escherichia coli and Bacillus subtilis, and find that paralogy relationships are insufficient to explain the global or local role observed for transcription factors (TFs) within regulatory networks. Our results provide a picture in which DNA-binding specificity, a molecular property that can be measured in different ways, is a predictor of the role of transcription factors. In particular, we observe that global regulators consistently display low levels of binding specificity, while displaying comparatively higher expression values in microarray experiments. In addition, we find a strong negative correlation between binding specificity and the number of co-regulators that help coordinate genetic expression on a genomic scale. A close look at several orthologous TFs,including FNR, a regulator found to be global in E. coli and local in B.subtilis, confirms the diagnostic value of specificity in order to understand their regulatory function, and highlights the importance of evaluating the metabolic and ecological relevance of effectors as another variable in the evolutionary equation of regulatory networks. Finally, a general model is presented that integrates some evolutionary forces and molecular properties,aiming to explain how regulons grow and shrink, as bacteria tune their regulation to increase adaptation.

Understanding the mechanisms by which transcriptional regulatory networks (TRNs) change through evolution is a fundamental problem.Here, we analyze this question using data from Escherichia coli and Bacillus subtilis, and find that paralogy relationships are insufficient to explain the global or local role observed for transcription factors (TFs) within regulatory networks. Our results provide a picture in which DNA-binding specificity, a molecular property that can be measured in different ways, is a predictor of the role of transcription factors. In particular, we observe that global regulators consistently display low levels of binding specificity, while displaying comparatively higher expression values in microarray experiments. In addition, we find a strong negative correlation between binding specificity and the number of co-regulators that help coordinate genetic expression on a genomic scale. A close look at several orthologous TFs,including FNR, a regulator found to be global in E. coli and local in B.subtilis, confirms the diagnostic value of specificity in order to understand their regulatory function, and highlights the importance of evaluating the metabolic and ecological relevance of effectors as another variable in the evolutionary equation of regulatory networks. Finally, a general model is presented that integrates some evolutionary forces and molecular properties,aiming to explain how regulons grow and shrink, as bacteria tune their regulation to increase adaptation.

Understanding the mechanisms by which transcriptional regulatory networks (TRNs) change through evolution is a fundamental problem. Here we analyze this question using data from Escherichia coli and Bacillus subtilis, finding that paralogy relationships are insufficient to explain the global or local role observed for transcription factors (TFs) within regulatory networks. Our results provide a picture in which DNA-binding specificity, a molecular property that can be measured in different ways, is a predictor of the role of transcription factors. In particular, we observe that global regulators consistently display low binding specificities, while displaying comparatively higher expression values in microarray experiments. In addition, in this work we find a strong negative correlation between binding specificity and the number of co-regulators which help coordinate genetic expression at a genomic scale. A close look at several orthologous TFs, including FNR, a regulator found to be global in E. coli and local in B. subtilis, confirms the diagnostic value of specificity in order to understand their regulatory function, and also highlights the importance of evaluating the metabolic and ecological relevance of effectors as another variable in the evolutionary equation of regulatory networks. Finally, a general model is presented that integrates some evolutionary forces and molecular properties, aiming to explain how regulons grow and shrink, as bacteria tune their regulation to increase adaptation.

The Lrp/AsnC family of transcriptional regulatory proteins is found in both archaea and bacteria. Members of the family influence cellular metabolism in both a global (Lrp) and specific (AsnC) manner, often in response to exogenous amino acid effectors. In the present study we have determined both the first bacterial and the highest resolution structures for members of the family. Escherichia coli AsnC is a specific gene regulator whose activity is triggered by asparagine binding. Bacillus subtilis LrpC is a global regulator involved in chromosome condensation. Our AsnC-asparagine structure is the first for a regulator¿effector complex and is revealed as an octameric disc. Key ligand recognition residues are identified together with a route for ligand access. The LrpC structure reveals a stable octamer supportive of a topological role in dynamic DNA packaging. The structures yield significant clues to the functionality of Lrp/AsnC-type regulators with respect to ligand binding and oligomerization states as well as to their role in specific and global DNA regulation.