Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Signal integration in bacterial two-component regulatory systems Alexander Y. Mitrophanov and Eduardo A. Groisman1 Department of Molecular Microbiology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, USA Two-component systems (TCSs) and phosphorelays are response regulator changes the biochemical properties key mediators of bacterial signal transduction. The sig- of its output domain, which can participate in DNA nals activating these systems promote the phosphorylat- binding and transcriptional control, perform enzymatic ed state of a response regulator, which is generally the activities, bind RNA, or engage in protein–protein inter- form that carries out specific functions such as binding actions (Gao et al. 2007). In addition to serving as phos- to DNA and catalysis of biochemical reactions. An phoryl donors, certain sensor kinases display phos- emerging class of proteins—termed TCS connectors— phatase activity toward their cognate phosphorylated modulate the output of TCSs by affecting the phosphor- regulators. ylation state of response regulators. TCS connectors use Phosphorelays are a more complex version of the TCS different mechanisms of action for signal integration, as in which a sensor kinase first transfers the phosphoryl well as in the coordination and fine-tuning of cellular group to a response regulator possessing the domain with processes. Present in both Gram-positive and Gram- the conserved aspartate but no output domain (Appleby negative bacteria, TCS connectors are critical for a vari- et al. 1996; Perraud et al. 1999). The response regulator ety of physiological functions including sporulation, subsequently transfers the phosphoryl group to a histi- competence, antibiotic resistance, and the transition to dine-containing phosphotransfer protein, and it is the stationary phase. latter protein that serves as a phosphodonor to the ter- minal response regulator, which possesses an output do- main mediating a cellular response (Fig. 1). In some phos- Free-living organisms modulate their gene expression phorelays, the sensor kinase and the response regulator patterns in response to environmental cues. This modu- lacking the output domain (and sometimes also the his- lation requires sensors to detect chemical and/or physi- tidine-containing phosphotransfer protein) are fused in a cal signals, and regulators to bring about changes in the single polypeptide (Appleby et al. 1996). levels of gene products. Certain cellular processes re- The vast majority of response regulators are active quire the integration of multiple signals into the deci- only when phosphorylated (Hoch 2000; Gao et al. 2007). sion to promote or inhibit the expression of a given gene Therefore, any condition or product that affects the product, which raises questions about the mechanisms phosphorylated state of a response regulator will impact used by different organisms to connect signal transduc- its ability to exert its biological functions. Conse- tion pathways and genetic regulatory circuits. quently, the output of a response regulator is determined In bacteria, extracellular signals are transduced into not only by the presence of the specific signals sensed by the cell predominantly by two-component systems its cognate sensor kinase but also by gene products that (TCSs) (Hoch 2000; Stock et al. 2000; Mascher et al. stimulate or inhibit its phosphorylation. Such products 2006; Gao et al. 2007). The prototypical TCS consists of can, in principle, target any one of the various steps lead- a sensor kinase that responds to specific signals by modi- ing to phosphorylation of the response regulator, includ- fying the phosphorylated state of a cognate response ing sensor kinase autophosphorylation, phosphotransfer regulator (i.e., the second component) (Fig. 1). Sensor ki- to the response regulator, dephosphorylation of a phos- nases are usually integral membrane proteins that auto- phorylated response regulator, and the activity of the phosphorylate from ATP at a conserved histidine residue output domain. The presence of multiple stages in a and then transfer the phosphoryl group to a conserved phosphorelay provides additional potential targets for aspartate in the response regulator. Phosphorylation of a control. TCS connectors (which for the sake of brevity will also be called connectors) are an emerging group of proteins [Keywords: Gene regulation; phosphorelay; sensor; two-component sys- that modulate the activity of sensor kinases and re- tem] sponse regulators at the post-translational level. Because 1Corresponding author. E-MAIL [email protected]; FAX (314) 747-8228. connector proteins are typically synthesized in response Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1700308. to signals that are different from those sensed by the GENES & DEVELOPMENT 22:2601–2611 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org 2601 Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Mitrophanov and Groisman Figure 1. Schematics of the proteins and do- mains that constitute TCSs (left) and phosphore- lays (right). The input domain of a sensor kinase responds to its signal by activating the autoki- nase domain, which autophosphorylates from ATP at a conserved histidine residue. The phos- phorylated sensor kinase interacts with the re- ceiver domain of the response regulator, which catalyzes the phosphoryl transfer to a conserved aspartate residue. Phosphorylation of the re- sponse regulator activates its output domain, which performs a specific biochemical function such as transcriptional regulation. A phosphore- lay contains, besides the sensor kinase and the terminal response regulator, an intermediate re- sponse regulator lacking an output domain and a His-containing phosphotransfer protein. In some phosphorelays, the phosphotransfer protein and/ or the intermediate response regulator is fused with the sensor kinase in a single polypeptide. cognate sensor, they often establish regulatory links be- limiting conditions (Piggot and Hilbert 2004). Because tween otherwise independent signal transduction path- sporulation is an energy-consuming process that be- ways (in other words, they “connect” a TCS to the sig- comes irreversible at an early stage (Dubnau and Losick nal(s) controlling a different regulatory system). Here we 2006), commitment to sporulation is tightly regu- describe the critical roles played by bacterial TCS con- lated and coordinated with other physiological func- nectors in a variety of cellular functions, including the tions. B. subtilis sporulation is governed by a phos- adaptation to nutrient-limiting conditions, sporulation, phorelay whereby five different sensor kinases—termed competence, antibiotic resistance, and the transition to KinA, KinB, KinC, KinD, and KinE—serve as phosphoryl stationary phase. We also discuss the distinct dynamic donors for the single-domain response regulator Spo0F properties of the regulatory circuits in which connectors (Piggot and Hilbert 2004). Spo0F then transfers the participate, and we examine how dissimilarities in the phosphoryl group to the histidine-containing phos- sequences of connectors or their genes’ promoters can photransfer protein Spo0B, which in turn transfers it to result in phenotypic differences among closely related the response regulator Spo0A, a DNA-binding protein bacterial species. that controls the expression of several genes, including those involved in sporulation (Fig. 2; Piggot and Hilbert 2004). TCS connectors use a variety of mechanisms to alter The Sda and KipI proteins modulate the levels of phos- response regulator output phorylated Spo0A (Spo0A-P), which constitutes the out- Connector proteins modulate the levels of the active put of the B. subtilis phosphorelay, by blocking auto- form of response regulators by affecting the various pos- phosphorylation of the sensor kinase KinA (Fig. 2; Wang sible steps that determine their phosphorylation state or et al. 1997; Burkholder et al. 2001). KinB is a possible their activity. Below, we present the various mecha- second target for inhibition by Sda (Burkholder et al. nisms of action adopted by connectors in the context of 2001). The sda gene is under transcriptional control of their physiological functions. the key replication initiation factor DnaA, and mutation of the dnaA gene leads to overexpression of Sda and in- hibition of sporulation (Burkholder et al. 2001). It was Inhibiting sensor kinase autophosphorylation proposed that conditions that affect replication initia- The Gram-positive soil bacterium Bacillus subtilis tion alter the level of active DnaA, thereby regulating forms a dormant spore when it experiences nutrient- sda through DnaA (Burkholder et al. 2001). Thus, the 2602 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Signal integration by bacterial systems Figure 2. TCS connectors can inhibit sensor autophosphorylation or promote dephosphorylation of phosphorylated re- sponse regulators. In the B. subtilis phos- phorelay, the sensor kinase KinA is acti- vated by an unknown signal, which results in autophosphorylation from ATP and subsequent phosphotransfer to the re- sponse regulator Spo0F. Spo0F passes on the phosphoryl group to the His-contain-