Research
Our research program seeks answers to a fundamental biological question:
How does an organism know when, where and for long to turn a gene on or off?
We address this question by investigating bacterial species
that establish intimate interactions with animal hosts.
Visit our Research page for more information.
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Intrinsic Negative Feedback Governs Activation Surge in Two-Component Regulatory Systems.
Bacterial two-component systems are a form of signal transduction that alters the behavior of an organism in response to changes in its surroundings. They consist of sensor kinase (SK) that modifies the phosphorylation of a cognate response regulator (RR). When experiencing inducing conditions, the levels of phosphorylated RR (RR-P) increase, peak and then decrease to a steady-state level that is ~25% of the maximum. ADP, which is endogenously generated from ATP during the autophosphorylation of the SK, simulates the SK's phosphatase activity towards RR-P. The resulting surge in RR-P levels is critical for a pathogen's ability to cause disease.
(A) In wild-type Salmonella, transcription of PmrA-activated genes is promoted during growth in low Mg2+ via the PhoP/PhoQ system, the PmrD protein and the PmrA/PmrB system, and in the presence of Fe3+ via the PmrA/PmrB system and independently of PhoP/PhoQ and PmrD. The PmrA protein represses transcription of the pmrD gene. (B) In wild-type E. coli, transcription of PmrA-activated genes is promoted in the presence of Fe3+ via the PmrA/PmrB system. The PmrD protein is produced in low Mg2+ in a PhoP-dependent manner but fails to activate the PmrA/PmrB system.
H. Deborah Chen, et al. PLoS Genet. 2011 July;7(7):e1002184
Limited distribution of Y. pestis and S. enterica PhoP-activated genes across the Enterobacteriaceae family.
A–B. Matrices of conservation scores (CS) of proteins encoded by the genes directly activated by PhoP in Y. pestis (A) and S. enterica (B). CS is the BlastP score of the closest homologue in a particular species divided by the BlastP score of the protein against itself. CS values (represented by colors) can range from 0 when no homolog or ortholog is detected in another species, to 1 when the closest homolog exhibits 100% amino acid identity. The phylogenetic relationships shown to the left of the figures are based on orthologous housekeeping genes present in all species. Note that branch lengths do not represent phylogenetic distances.
Differential control mediated by orthologous transcription factors.
Depiction of three related bacterial species (termed A, B and C) sharing an ancestral transcription factor, which regulates variable gene sets in the three organisms. Only two target genes (yellow and orange arrows) are shared among the three species. The remaining target genes are regulated by the transcription factor in only one of the species because the target gene(s) is species-specific (green and lavender arrows) or because only one of the species harbors binding sites for the transcription factor in the promoter region of genes shared across species (brown and blue arrows.) The shared target genes, constituting the core regulon, have two main roles: to cope with the environmental change that activates the regulon, and to control the amount of the active form of the transcription factor. By contrast, the species-specific targets aid each species to proliferate in the particular niches in which they live.
Genetic regulatory architectures with positive feedback.
(a) Prototypic two-component signal transduction system. X – regulator protein, Y – sensor protein; x and y denote the corresponding genes, which are part of the same operon. Phosphorylated regulator binds with the genes’ autoregulated promoter and activates transcription. (b) The modular model of two-component signal transduction (redrawn from Ref. 6 with modifications). The phosphorylation module determines the concentration of X-P (phosphorylated regulator) as a function of the total sensor and regulator concentrations and the intensity of the activating stimulus. The autoregulation module defines the total sensor and regulator concentrations given the concentration of phosphorylated regulator. (c) A dynamic model of positive autoregulation introduced in Ref. 4. The regulator X (in phosphorylated form) can directly activate expression of its own gene.
Expression of the Salmonella Mg2+ Transporter Gene mgtA Is Regulated by the PhoP/PhoQ System, the Rob Protein and by the mgtA LR.
When the sensor PhoQ detects low extracytoplasmic Mg2+, acid pH or antimicrobial peptides, it promotes phosphorylation of the PhoP protein, which binds to the mgtA promoter resulting in transcription initiation. Rob promotes mgtA transcription in response to a yet unidentified signal. Transcription elongation into the mgtA CR is regulated by the mgtA leader via a Mg2+-sensing riboswitch and by translation of an 18-codon proline-rich ORF designated mgtL. The mgtL ribosome binding site is denoted by RBS adjacent to a black line. Positions and sequences of stop codon mutations in the strains used in the experiments presented in Figure 4 are denoted below the linear mgtL RNA sequence. High Mg2+ and high proline conditions promote formation of stem-loop B, hindering transcription elongation into the mgtA CR. Low Mg2+ and/or low proline favor formation of stem-loop C, resulting in transcription of the mgtA CR. See also Figure S1.
A Bacterial mRNA Leader that Employs Different Mechanisms to Sense Disparate Intracellular Signals.
The leader region of many bacterial mRNAs has the ability to form mutually exclusive secondary structures that determine whether transcription will continue into the adjacent coding region. Which secondary structure forms is governed by the binding of specific metabolites, ions, tRNAs, or proteins to the LR, by pausing of RNA polymerase at particular sequences within the LR and by translation of short open reading frames (ORFs) located in the leader RNA. The leader RNA of the Mg2+ transporter gene mgtA of Salmonella enterica provides a singular example of a leader RNA that utilizes different mechanisms to sense two different signals. It functions as a Mg2+-responding riboswitch and as a sensor for cytoplasmic proline via the coupling/uncoupling of transcription of the mgtA leader RNA and translation of short ORF rich in proline codons located in the mgtA leader.
Scatter plot combining the ranking of ancestry with the CG-content (Bioinformatics Toolbox, Matlab 2007b) of the 23 proteins in Salmonella (F statistic P-value < 0.0005). The ranking of ancestry is defined as the order in which genes were incorporated into the clusters plotted in (A) (i.e. order among conserved ORF sequences across species, see Supporting information). Colours correspond to promoter architectures (yellow: I, blue: II, brown: III, magenta: IV, cyan: V. The empty square indicates a reclassification of the orgB promoter).
Subclasses of the PhoP box consensus sequence (submotifs).
These subclasses were identified based on footprinting data (Table 1; Fig. S3) as described in Harari et al. (2010) and Zwir et al. (2005), where one sequence can belong to more than one cluster, and a subset of sequences can be hierarchically organized based on their specificity and sensitivity. A. Submotifs corresponding to the canonical direct repeat consensus sequence (S1). Three different subpatterns were identified within this class. B. Submotif S2 corresponding to a conserved variant in the first repeat sequence. C. Submotif S3 corresponding to a conserved variant in the second repeat sequence.
The sensor PhoQ responds to extracytoplasmic low Mg2+, acidic pH and antimicrobial peptides by promoting phosphorylation of the PhoP protein, which binds to the mgtCBR promoter, resulting in transcription initiation. Acidic pH also produces a proton gradient across the inner membrane, resulting in higher intracellular ATP levels. Intracellular ATP levels control transcription elongation into the mgtCBR coding regions by affecting the coupling/uncoupling of transcription of the mgtCBR leader and translation of the short ORF mgtM, which affects the formation of alternative stem-loops A or B. The mgtM ribosome-binding site is underlined. Positions and sequences of stop codon mutations or nucleotide substitutions in the strains used in the experiments presented in c and d are indicated and denoted below the mgtM sequence.
