You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: The Architecture of a Prototypical Bacterial Signaling Circuit Enables a Single Point Mutation to Confer Novel Network Properties}
Article:
Abstract:
Even a single mutation can cause a marked change in a protein's properties. When the mutant protein functions within a network, complex phenotypes may emerge that are not intrinsic properties of the protein itself. Network architectures that enable such dramatic changes in function from a few mutations remain relatively uncharacterized. We describe a remarkable example of this versatility in the well-studied PhoQ/PhoP bacterial signaling network, which has an architecture found in many two-component systems. We found that a single point mutation that abolishes the phosphatase activity of the sensor kinase PhoQ results in a striking change in phenotype. The mutant responds to stimulus in a bistable manner, as opposed to the wild-type, which has a graded response. Mutant cells in on and off states have different morphologies, and their state is inherited over many generations. Interestingly, external conditions that repress signaling in the wild-type drive the mutant to the on state. Mathematical modeling and experiments suggest that the bistability depends on positive autoregulation of the two key proteins in the circuit, PhoP and PhoQ. The qualitatively different characteristics of the mutant come at a substantial fitness cost. Relative to the off state, the on state has a lower fitness in stationary phase cultures in rich medium( LB). However, due to the high inheritance of the on state, a population of on cells can be epigenetically trapped in a low-fitness state. Our results demonstrate the remarkable versatility of the prototypical two-component signaling architecture and highlight the tradeoffs in the particular case of the PhoQ/PhoP system.
Introduction:
A few mutations can lead to significant changes in a protein's functional properties. Examples include mutations that change the absorption and emission spectra of a fluorescent protein[1], the substrate specificity of an enzyme[2], or the allosteric control of a transcription factor[3]. In all of these examples, the change in phenotype can be directly traced to modifications in intrinsic properties of the protein. However, networks of interacting proteins can have system-level characteristics that bear a complex relationship to the intrinsic properties of the component molecules[4]. This complexity makes some network architectures inherently versatile, with different networks that share the same architecture exhibiting qualitatively different system-level behavior[5]. It remains a challenge to identify aspects of network architectures that promote versatility and permit novel properties to emerge by a few mutations to network components. In this study, we demonstrate the versatility of the E. coli PhoQ/PhoP system. We show that a single point mutation in the histidine kinase PhoQ produces a striking change in the properties of the circuit. The PhoQ/PhoP system, which has an architecture found in many bacterial two-component signaling systems[6], responds to a variety of environmental conditions such as low Mg2+[7], low pH[8], and the presence of cationic antimicrobial peptides[9], and controls transcription of a large set of genes[10]. The histidine kinase PhoQ senses these signals and modulates the phosphorylation level of the response regulator PhoP( PhoP-P), which functions as a transcription factor. PhoQ autophosphorylates and then transfers the phosphoryl group to PhoP, but also acts as a phosphatase, catalyzing PhoP-P dephosphorylation[11]. This bifunctional design, which is shared among many two-component systems, affects various properties of the system, including buffering the input-output relationship of the system to changes in histidine kinase and response regulator concentrations, and suppression of cross-talk[12]–[15]. The PhoQ/PhoP system is also autoregulated, that is, transcription of the phoPphoQ operon is activated by PhoP-P. Autoregulation is another common feature of many two-component systems[6] and is a mechanism for ultrasensitive response to stimulus without the need for cooperativity[16] as well as “learning” behaviors where prior exposure to stimulus improves response times to subsequent stimulating conditions[17],[18]. In the case of the PhoQ/PhoP system, autoregulation improves the dynamic range of the network output at high stimulus[13] and also gives rise to a surge in transcription upon activation[19]. The wild-type PhoQ/PhoP system responds to external stimulus in a graded rather than an all-or-none manner, with increasing stimulus( lower Mg2+ concentration) resulting in a higher mean response[13]. Moreover, the response of a population of cells is unimodal, with the PhoP-P levels of individual cells( inferred from transcriptional reporters) clustered around the population mean[13]. In this study, we show that a single point mutation that abolishes phosphatase activity in PhoQ produces a dramatic change in phenotype. The mutation, which produces a T281R substitution in PhoQ, results in bistability with mutants persisting in morphologically distinct OFF and ON states for many generations. We find that the architectural features of the network that allow the mutant to exhibit bistability are shared among many two-component systems. For PhoQ( T281R) mutants, however, the bistable phenotype comes at a fitness cost. We find that a population of cells can be epigenetically trapped in a low-fitness state.
Discussion:
We have demonstrated that a single point mutation, causing a T281R substitution in the histidine kinase PhoQ, results in a remarkable change in phenotype, with cells exhibiting bistability/bimodality and an inverted response to[Mg2+]. Previous modeling work indicates that two-component systems have the potential to exhibit bimodal behavior as a result of stochastic fluctuations in molecular components[16],[20],[21] as well as bistability[26],[27]. Bistability has been observed experimentally in the MprB/MprA two-component system[28], although additional feedback loops not present in simple two-component signaling architectures may be responsible for the bistability in that system[29]. Beyond two-component systems, the response of the lac operon to certain gratuitous inducers is another setting where bistability and strong epigenetic inheritance has been observed in bacteria[30],[31]. Like the lac operon, we find the OFF and ON states in PhoQ( T281R) can be inherited over many generations. However, we have not been able to find conditions where ON cells can be deterministically converted to an OFF state. Such irreversibility has been seen in the development of Xenopus oocytes[32] and is more akin to transient or terminal differentiation seen during bacterial competence or sporulation. Both competence and sporulation are highly regulated phenomena[33] and neither is, strictly speaking, a manifestation of a simple positive-feedback based bistable switch[34],[35]. In this sense, irreversibile bistability is a distinctive feature of the PhoQ( T281R) network among characterized two-component signaling systems. While bistability and noise-induced bimodality are alternate explanations for the observed behavior of the PhoQ( T281R) network, it may be difficult to differentiate between these mechanisms experimentally. First, in our simulations, we observe noise-induced bimodality only for monostable parameters close to the true bistable regime( Text S1). Thus, distinguishing between these mechanisms would likely require careful measurement of kinetic parameters in vivo, which can be challenging even for simple networks. Second, for the mechanism based on noise-induced biomodality, if the timescale for conversion from OFF to ON state in the bimodal regime is tens of generations, then any small fitness difference between the two states can become relevant and even lead to the emergence of bistability, as has been shown in a synthetic circuit[36]. What are the architectural features of the PhoQ/PhoP circuit that enable the T281R mutation to produce such unusual properties? The modeling presented here reveals that the mutant network can readily show bistable behavior when both the kinase and phosphatase activities of the histidine kinase are low( Text S1). A point mutation that simultaneously affects both functions of the histidine kinase can produce a bistable response, as is the case with the T281R mutation, where the kinase activity is low and the phosphatase activity is not detectable. Modeling also indicated that autoregulation of both the response regulator and histidine kinase is required for bistability( Figure 5B). Thus, since phoP and phoQ are organized as an operon that is driven by a PhoP-P responsive promoter, the architecture of the PhoQ/PhoP network( Figure 1A) is poised to show bistability upon introduction of the T281R mutation in PhoQ. Given that bifunctional histidine kinases and autoregulation are common themes in two-component signaling[6], mutations decreasing phosphatase activity in other histidine kinases may similarly lead to bistable behavior. Since our model does not explicitly incorporate the role of[Mg2+] as an input signal, we can only speculate on the potential mechanism for the inversion of[Mg2+]-sensitivity in the mutant. In terms of our model, the inversion can be explained if higher[Mg2+] increases the kinase rate of PhoQ( T281R) or increases the maximal expression rate of the phoPphoQ operon( potentially through a PhoP-P independent mechanism). Both of these effects could be masked in the wild-type network. For example, an increase in kinase rate at high[Mg2+] could be accompanied by a much larger increase in phosphatase rate in PhoQ( WT) resulting in the observed repression of PhoP-P levels under these conditions. Any increase in transcriptional activity is likely to be unnoticed in the wildtype as the output for the wild-type circuit is relatively insensitive to PhoP and PhoQ protein levels for most ranges of magnesium concentration[13]. We should also note that the reversal of input sensitivity may be an idiosyncrasy of the PhoQ/PhoP system and not a general feature of phosphatase mutants of bifunctional histidine kinases. The phoQ( T281R) mutant strain exhibits phenotypic hysteresis, that is, the state of a population depends strongly on the culture history( Figure 1D and 2B). OFF cells retain their state in exponential phase, but prime to the ON state upon passage through stationary phase in minimal medium with low[Mg2+]( Figure 2B). In the lac operon, slow growth rate has been shown to increase the transition probability from the uninduced to induced state through the accumulation of inducer and the permease protein LacY[37]. A reduction in growth rate of the phoQ( T281R) strain can similarly cause PhoP and PhoQ to accumulate because of residual expression of the phoPphoQ operon. In addition, PhoP-P would be expected to accumulate from PhoQ( T281R) kinase activity and from the lack of a specific phosphatase. Either mechanism can account for the phenomenon of priming. Is it possible for strains with wild-type phoQ to show bistability? The wild-type PhoQ/PhoP system responds to lowering Mg2+ concentrations in a unimodal, graded manner[13]. As long as the loss of PhoP-P due to the phosphatase activity of PhoQ( WT) dominates over the reduction in PhoP-P concentration due to growth-mediated dilution, the network is monostable( Text S1 and ref.[13]). It is conceivable that under highly activating conditions, the phosphatase activity of PhoQ( WT) is sufficiently low, in effect, phenocopying the phosphatase defect in phoQ( T281R). Under these circumstances, our analysis( Text S1) suggests that bistability is possible provided that( 1) enough PhoP-P can be produced to begin to saturate the PhoP-P mediated feedback expression of the phoPQ operon and( 2) the constitutive expression of the operon is sufficiently low( Figure S1B). However, at a Mg2+ concentration of 100 µM, which is an activating stimulus for PhoQ( WT), condition( 1) is not satisfied in the wild-type network[38]. It is, therefore, possible that the wild-type circuit has evolved to always maintain a significant level of phosphatase activity to avoid bistability. The phenotypic heterogeneity associated with bistability can improve fitness in fluctuating or unpredictable environments[39],[40]. Bistability also provides a mechanism for storing information about past environmental conditions, and the persistence of this information can be influenced by the network architecture[41]. In the case of phoQ( T281R), however, bistability comes at a fitness cost because of its irreversible nature. Long-term culture experiments in LB showed that a population of ON cells could be trapped in this low-fitness state. In principle, epigenetic trapping can also occur in a reversible system, but in that case there would be environmental conditions in which the population can deterministically transition to the high-fitness state. In an irreversible system, rare stochastic transitions followed by enrichment due to selection are the only means of escaping from the trap. Interactions between multiple levels of organization in biological systems can result in unexpected or counter-intuitive phenomena. For example, noise at the molecular level can produce bimodal outcomes in a genetic network that is monostable when analyzed as a deterministic system[42]. Likewise, bistability can emerge in a monostable network because of the influence of the output of the network on the growth rate of the organism[36]. In the case of phoQ( T281R), we find that a bistable genetic network gives rise to a stable and a metastable mode since one of the stable states of the network is inherited efficiently, but has a fitness disadvantage. The phoQ( T281R) mutation also perfectly illustrates a recent suggestion by Kitano that biological networks may be more sensitive to ‘fail-on’ failures where components function in unexpected ways than to ‘fail-off’ failures where components do not function at all or are removed[43]. While a phoQ deletion would be unresponsive to Mg2+ levels, it would not show bistability or epigenetic trapping. In contrast, a single point mutation in the robust PhoQ/PhoP signaling module results in a phosphatase-deficient PhoQ protein that gives rise to bistability and an ON state with pleiotropic alterations in cell physiology.
A mutation can cause significant changes to a protein's function . Since proteins often act together in genetic circuits to control various cellular processes , mutant proteins can lead to unexpected consequences for system-level behavior . In this study , we describe a remarkable example of this phenomenon in a mutant of a well-studied bacterial circuit . PhoQ and PhoP are the primary regulatory proteins in a circuit that responds to low magnesium . The wild-type ( unmutated ) network responds to environmental signals in an analog or graded manner . In contrast , the mutant responds to signals in an OFF-or-ON or digital fashion . Moreover , the distribution of OFF and ON cells is strongly influenced by how cells were cultured in the past . These remarkable changes can be traced to features of the wiring diagram of the PhoQ/PhoP circuit . Since these features are shared among a broad class of bacterial signaling circuits , we suggest that other circuits may show similar remarkable properties when mutated .