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We have evolved a robust two-component signal transduction pathway from a sensor kinase (SK) and non-partner response regulator (RR) that show weak cross-talk in vitro and no detectable cross-talk in vivo in wild-type strains. The SK, CpxA, is bifunctional, with both kinase and phosphatase activities for its partner RR. We show that by combining a small number of mutations in CpxA that individually increase phosphorylation of the non-partner RR OmpR, phosphatase activity against phospho-OmpR emerges. The resulting circuit also becomes responsive to input signal to CpxA. The effects of combining these mutations in CpxA appear to reflect complex intragenic interactions between multiple sites in the protein. However, by analyzing a simple model of two-component signaling, we show that the behavior can be explained by a monotonic change in a single parameter controlling protein-protein interaction strength. The results suggest one possible mode of evolution for two-component systems with bifunctional SKs whereby the remarkable properties and competing reactions that characterize these systems can emerge by combining mutations of the same effect.

In a developmental strategy designed to efficiently exploit and colonize sparse oligotrophic environments, Caulobacter crescentus cells divide asymmetrically, yielding a motile swarmer cell and a sessile stalked cell. After a relatively fixed time period under typical culture conditions, the swarmer cell differentiates into a replicative stalked cell. Since differentiation into the stalked cell type is irreversible, it is likely that environmental factors such as the availability of essential nutrients would influence the timing of the decision to abandon motility and adopt a sessile lifestyle. We measured two different parameters in nutrient-limited chemostat cultures, biomass concentration and the ratio of nonstalked to stalked cells, over a range of flow rates and found that nitrogen limitation significantly extended the swarmer cell life span. The transcriptional profiling experiments described here generate the first comprehensive picture of the global regulatory strategies used by an oligotroph when confronted with an environment where key macronutrients are sparse. The pattern of regulated gene expression in nitrogen- and carbon-limited cells shares some features in common with most copiotrophic organisms, but critical differences suggest that Caulobacter, and perhaps other oligotrophs, have evolved regulatory strategies to deal distinctly with their natural environments. We hypothesize that nitrogen limitation extends the swarmer cell lifetime by delaying the onset of a sequence of differentiation events, which when initiated by the correct combination of external environmental cues, sets the swarmer cell on a path to differentiate into a stalked cell within a fixed time period. doi: 10.1128/JB.01240-09

In Caulobacter crescentus, progression through the cell cycle is governed by the periodic activation and inactivation of the master regulator CtrA. Two phosphorelays, each initiating with the histidine kinase CckA, promote CtrA activation by driving its phosphorylation and by inactivating its proteolysis. Here, we examined whether the CckA phosphorelays also influence the downregulation of CtrA. We demonstrate that CckA is bifunctional, capable of acting as either a kinase or phosphatase to drive the activation or inactivation, respectively, of CtrA. By identifying mutations that uncouple these two activities, we show that CckA's phosphatase activity is important for downregulating CtrA prior to DNA replication initiation in vivo but that other phosphatases may exist. Our results demonstrate that cell cycle transitions in Caulobacter require and are likely driven by the toggling of CckA between its kinase and phosphatase states. More generally, our results emphasize how the bifunctional nature of histidine kinases can help switch cells between mutually exclusive states. doi: 10.1128/JB.00992-09

Vibrio cholerae causes the disease cholera and inhabits aquatic environments. One key factor in the environmental survival of V. cholerae is its ability to form matrix-enclosed, surface-associated microbial communities known as biofilms. Mature biofilms rely on Vibrio polysaccharide to connect cells to each other and to a surface. We previously described a core regulatory network, which consists of two positive transcriptional regulators, VpsR and VpsT, and a negative transcriptional regulator HapR, that controls biofilm formation by regulating the expression of vps genes. In this study, we report the identification of a sensor histidine kinase, VpsS, which can control biofilm formation and activates the expression of vps genes. VpsS required the response regulator VpsR to activate vps expression. VpsS is a hybrid sensor histidine kinase that is predicted to contain both histidine kinase and response regulator domains, but it lacks a histidine phosphotransferase (HPT) domain. We determined that VpsS acts through the HPT protein LuxU, which is involved in a quorum-sensing signal transduction network in V. cholerae. In vitro analysis of phosphotransfer relationships revealed that LuxU can specifically reverse phosphotransfer to CqsS, LuxQ, and VpsS. Furthermore, mutational and phenotypic analyses revealed that VpsS requires the response regulator LuxO to activate vps expression, and LuxO positively regulates the transcription of vpsR and vpsT. The induction of vps expression via VpsS was also shown to occur independent of HapR. Thus, VpsS utilizes components of the quorum-sensing pathway to modulate biofiim formation in V. cholerae.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.04.040 Byline: Jeffrey M. Skerker (1), Barrett S. Perchuk (1), Albert Siryaporn (2), Emma A. Lubin (1), Orr Ashenberg (1), Mark Goulian (2)(3), Michael T. Laub (1) Keywords: SIGNALING; MICROBIO Abstract: Two-component signal transduction systems are the predominant means by which bacteria sense and respond to environmental stimuli. Bacteria often employ tens or hundreds of these paralogous signaling systems, comprised of histidine kinases (HKs) and their cognate response regulators (RRs). Faithful transmission of information through these signaling pathways and avoidance of detrimental crosstalk demand exquisite specificity of HK-RR interactions. To identify the determinants of two-component signaling specificity, we examined patterns of amino acid coevolution in large, multiple sequence alignments of cognate kinase-regulator pairs. Guided by these results, we demonstrate that a subset of the coevolving residues is sufficient, when mutated, to completely switch the substrate specificity of the kinase EnvZ. Our results shed light on the basis of molecular discrimination in two-component signaling pathways, provide a general approach for the rational rewiring of these pathways, and suggest that analyses of coevolution may facilitate the reprogramming of other signaling systems and protein-protein interactions. Author Affiliation: (1) Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA (2) Department of Physics, University of Pennsylvania, Philadelphia, PA 19104, USA (3) Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA Article History: Received 7 February 2008; Revised 4 April 2008; Accepted 15 April 2008 Article Note: (miscellaneous) Published: June 12, 2008