All organisms respond to changes in their environment by modifying their behavior. We are interested in identifying the specific signals that denote a given environment, the nature of the sensors that detect such signals, and how the sensors transmit this information to the regulators, implementing a response that enables the organism to survive and prosper in the new condition.
We investigate the gastroenteritis- and typhoid fever–causing Salmonella enterica and the gut symbiotic bacteria Escherichia coli and Bacteroides thetaiotaomicron. We have been examining both protein sensors that detect extracytoplasmic signals and RNA sensors that monitor cytoplasmic metabolites and ions. These investigations led to the discovery of the first signal transduction systems that sense extracytoplasmic magnesium and ferric iron and to the identification of the first leader mRNAs that respond to cytoplasmic magnesium, proline, and ATP levels.
Our research is helping us understand how bacteria can integrate multiple signals into a cellular response, the mechanisms by which a given signal can elicit distinct responses from co-regulated targets, and the genetic basis for the phenotypic differences that distinguish closely related bacterial species, such as a pathogen and a symbiont. Moreover, our explorations are revealing the biochemical function of novel proteins, the genetic control of virulence factors, and the molecular basis for Mg2+ homeostasis.
Signal Transduction by Two-Component Regulatory Systems
Bacteria often rely on two-component regulatory systems to modulate their gene expression programs when there is a change in their surroundings. Two-component systems typically consist of an integral membrane sensor and a DNA-binding regulator. Sensors respond to the presence of their specific signal(s) by modifying the phosphorylation state of their cognate regulatory proteins, which are typically DNA-binding proteins whose affinity for their DNA targets is enhanced upon phosphorylation.
We have been investigating the biology of a pair of two-component systems in Salmonella: PhoP/PhoQ, which governs virulence and adaptation to Mg2+-limiting environments, and PmrA/PmrB, which controls the expression of proteins that modify the lipopolysaccharide, which constitutes the outermost layer of the bacterial outer membrane. We determined that when Salmonella are shifted to inducing conditions for these systems, the levels of phosphorylated regulator change dynamically, resulting in a surge of activity before the system reaches steady state. This is surprising because the changes in phosphorylated regulator take place under constant inducing conditions. The surge of phosphorylated PhoP protein is critical for Salmonella's ability to cause disease because a strain engineered to reach the same steady state monotonically (i.e., without the surge) is attenuated for virulence. These results demonstrate that organisms are sensitive not only to the particular activation level achieved in response to a given signal but also to how that activation level is reached.
Bacteria must tightly control the levels of phosphorylated regulator molecules. We identified three feedback mechanisms that modulate the output of two-component systems. First, there is positive feedback on transcription of the sensor and regulator genes. Second, intrinsic negative feedback enables a sensor to switch from acting (or functioning as) an autokinase and phosphotransferase to being a phosphatase, removing a phosphate group on the phosphorylated regulator. These two feedback mechanisms are responsible for the surge in the phosphorylated regulator levels described above. Finally, we uncovered a dynamic reciprocal feedback mechanism by which gene products under the control of the regulator PmrA modify the lipopolysaccharide to limit access to the signal that activates its cognate sensor PmrB.
Recently, we have been exploring an unusual class of two-component systems in the gut symbiont B. thetaiotaomicron in which the sensor and regulator proteins are fused into a single polypeptide. These hybrid two-component systems govern the import and breakdown of polysaccharides that are part of the mammalian diet. These investigations are revealing how the rate of polysaccharide utilization modulates the activity of the hybrid two-component system controlling the production of polysaccharide utilization genes.
Differential Gene Control by Leader mRNAs
Regulatory proteins often control expression of multiple genes, which typically encode products that are required in different amounts, under particular circumstances, or for specific times. This raises the question: How does an organism achieve differential expression of targets co-regulated by a given transcription factor? We have been addressing this question both by analyzing the architecture of promoters controlled by the PhoP protein and by exploring regulatory inputs mediated by the leader mRNA regions.
We determined that the promoter architecture (i.e., the number, location, and orientation of the PhoP-binding sites) of PhoP-activated genes ancestral to enteric bacteria is different from those found in promoters driving transcription of PhoP-activated genes that were horizontally acquired by Salmonella. The promoter architectures are responsible for the distinct expression behavior of PhoP-activated genes when Salmonella experience inducing conditions for the sensor PhoQ.
We established that the leader regions of certain PhoP-activated transcripts act as sensing devices that enable Salmonella to differentially control expression of individual PhoP-activated genes. For example, the leader region of the PhoP-activated Mg2+ transporter mgtA gene functions as a Mg2+ sensor that determines whether transcription continues into the mgtA-coding region and the MgtA protein is made. The Mg2+ sensor in the mgtA mRNA enables bacteria to exert separate control over determinants governing cytoplasmic Mg2+ homeostasis (i.e., a Mg2+ transporter) versus extracytoplasmic Mg2+ homeostasis (i.e., enzymes that modify Mg2+-binding sites in the cell envelope) when these determinants are all transcriptionally regulated by the PhoP/PhoQ system. In addition, the leader region of the PhoP-activated mgtCBR operon responds to changes in the levels of cytoplasmic ATP and proline by modulating transcription of the associated coding regions. The mgtCBR operon specifies the virulence protein MgtC, the Mg2+ transporter MgtB, and the regulatory peptide MgtR. The ability of the mgtCBR leader to respond to ATP is critical for Salmonella's ability to cause disease.
The Functions of Virulence and Antivirulence Genes
Pathogens have genes that enable them to gain access to and proliferate in host tissues. Paradoxically, pathogens can also have genes that actually decrease virulence. That is, bacteria retain genes that, upon inactivation, render pathogens more virulent. Surprisingly, the regulatory protein PhoP promotes expression of both virulence and antivirulence genes in Salmonella.
We have been exploring the targets and functions of virulence and antivirulence genes. We have uncovered novel ways by which virulence proteins enable Salmonella to withstand the microbicidal conditions presented by host phagocytic cells. We have also found that antivirulence genes can modify the host response to bacterial gene products and alter the kinetics of a natural infection.
Cumulatively, our investigations are revealing the logic behind the spatial and temporal expression behavior of genes targeted by a regulatory protein. Furthermore, they are revealing that bacteria have the ability to detect and respond to unexpected signals.
Grants from the National Institutes of Health provided partial support for some of these projects.
As of February 27, 2013