The Sensory Histidine Kinases TorS and EvgS Tend to Form Clusters in Escherichia coli Cells

Vaknin A (2013) The Sensory Histidine Kinases TorS and EvgS Tend to Form Clusters in Escherichia coli Cells . PLoS ONE 8(10): e77708. doi:10.1371/journal.pone.0077708 The Sensory Histidine Kinases TorS and EvgS Tend to Form Clusters in Escherichia coli Cells Erik Sommer 0 Moriah Koler 0 Vered Frank 0 Victor Sourjik 0 Ady Vaknin 0 Christopher V. Rao, University of Illinois at Urbana-Champaign, United States of America 0 1 Department of Molecular Biology, University of Heidelberg , Heidelberg, Germany , 2 Racah Institute of Physics, the Hebrew University , Jerusalem , Israel Microorganisms use multiple two-component sensory systems to detect changes in their environment and elicit physiological responses. Despite their wide spread and importance, the intracellular organization of two-component sensory proteins in bacteria remains little investigated. A notable exception is the well-studied clustering of the chemoreceptor-kinase complexes that mediate chemotaxis behaviour. However, these chemosensory complexes differ fundamentally from other systems, both structurally and functionally. Therefore, studying the organization of typical sensory kinases in bacteria is essential for understanding the general role of receptor clustering in bacterial sensory signalling. Here, by studying mYFP-tagged sensory kinases in Escherichia coli, we show that the tagged TorS and EvgS sensors have a clear tendency for self-association and clustering. These sensors clustered even when expressed at a level of a few hundred copies per cell. Moreover, the mYFP-tagged response regulator TorR showed clear TorS-dependent clustering, indicating that untagged TorS sensors also tend to form clusters. We also provide evidence for the functionality of these tagged sensors. Experiments with truncated TorS or EvgS proteins suggested that clustering of EvgS sensors depends on the cytoplasmic part of the protein, whereas clustering of TorS sensors can be potentially mediated by the periplasmic/transmembrane domain. Overall, these findings support the notion that sensor clustering plays a role in bacterial sensory signalling beyond chemotaxis. - Funding: This work was supported by the GIF grant I-909-54.13/2006, the ERC advanced grant 294761, and the Minerva center for bio-hybrid complex systems. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Two-component (TC) sensory systems are widespread in bacteria. Bacterial cells use these systems to sense environmental stimuli and elicit an appropriate adaptive cellular response [15]. A canonical TC system consists of two proteins: a sensory histidine kinase (HK) and a response regulator (RR). The sensory kinases are mostly membranebound receptors that sense different cues in the environment and communicate this information to their cytoplasmic kinase domain to control its rate of auto-phosphorylation at a conserved histidyl residue. The phosphoryl group can be then transferred to an aspartyl residue of the cognate response regulator. The response regulator protein is typically a transcription factor consisting of two domains: a receiver or regulatory domain that is phosphorylated by the kinase and an output domain that binds DNA [6,7]. The phosphorylation state of the receiver domain modifies the affinity of the binding domain for the DNA and thereby regulates expression of specific genes to elicit cellular responses. Various modifications of this scheme are found. For example, some sensors have dual function as kinase and phosphatase [8], which has been considered as a mechanism to generate a robust output [9,10]. Other systems, including the TorS and EvgS sensors, have multiple phosphotransfer steps within the sensor or between additional cytoplasmic proteins [11]. A unique member of the two-component family is the chemosensory system [12,13], which presumably evolved from the basic two-component scheme [2,14]. The chemosensory system controls the swimming behaviour of the bacterium with great sensitivity, large dynamic range, and fast response time [15]. In the chemosensory system, the sensing and the kinase functions are partitioned between two separated proteins: a transmembrane receptor and an associated cytoplasmic histidine kinase. The chemoreceptor/kinase complexes, together with a linker protein CheW, form tight hexagonal arrays, or clusters, located at the cell poles or along the cell body [1619]. Allosteric interactions in clusters lead to nonlinearity between the input signal and the output kinase activity, allowing amplification and integration of signals and increased dynamic range [14,2023]. However, while the chemotaxis system controls cell motility, most two-component systems mediate adaptive responses that involve gene expression. Thus, during its evolution, the chemotaxis system was subjected to functional constraints different from those faced by most two-component systems, and receptor clustering in chemotaxis might be a consequence of some of these unique functional constrains. Polar localization of sensory kinases was also demonstrated in the bacteria Caulobacter crescentus [24] and Xanthomonas campestris [25]. In the former, however, clustering appears to serve a highly specialized function in the asymmetric cell division. In E. coli, clustering of the DcuS and CitA sensory kinases [26,27] and the cytoplasmic response regulators OmpR and PhoP [28,29] have been reported. Nevertheless, the physical organization of most sensors remains unclear [30]. Investigating the cellular organization of the canonical sensory kinases is essential not only for understanding the functions of these individual systems but also for getting fundamental insights into the role of clustering in sensory signaling. Here we performed a systematic in-vivo study of physical associations and clustering of sensory kinases in E. coli, observing prominent tendency for clustering of the TorS and EvgS sensors. Results and Discussion Preliminary screen for self-association of sensory kinases in E. coli We constructed a plasmid library of C-terminal fusions of monomeric yellow fluorescent protein EYFPA206K (mYFP) to all 27 of the canonical transmembrane sensory kinases in E. coli. The cytoplasmic kinases CheA and NtrB and the plasmid-born sensor PcoS were not included in this study. We subsequently excluded the fusions to ArcB and RstB from further analysis since immunobloting analysis using an antibody against mYFP indicated that these fusions were highly degraded. The level of protein degradation of all other receptor fusions used in this study and their expression levels used for the imaging experiments are shown in Table S1. We have used fluorescence images and homo-FRET measurements [22,31] of these tagged sensors expressed in wild-type E. coli strain MG1655 to screen for sensors with potentially higher (...truncated)