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Extracytoplasmic Function σ Factors Come of Age Print E-mail

 

The extracytoplasmic function σ factors provide bacteria a third mechanism for responding to extracellular stimuli

Anna Staroń and Thorsten Mascher

Anna Staroń is a graduate student in the group of Thorsten Mascher, who is a professor at the Ludwig-Maximilians- University Munich, Dept. Biology I, Microbiology, D-82152 Planegg- Martinsried, Germany.

Summary

● Bacterial signal transduction involves one- and two-component systems and extracytoplasmic function σ factors.
● With its simple design, the one-component system is the most widely distributed type of bacterial signal transduction system.
● The design of two-component systems simplifies transmembrane signaling and also enables many modifications, including amplification and integration of different signals.
● The activity of ECF σ-factors is regulated by different mechanisms through a diverse set of anti-σ molecules.
● Although the two-component system and ECF σ factors are phylogenetically and biochemically unrelated, they share several mechanistic analogies while differing in the way their respective two-protein components communicate with each other.


One important lesson learned from the field of comparative genomics is that microbial genomes are modular. Genes encoding proteins of related functions are often encoded in one or more operons that are located adjacent to each other on the bacterial chromosome. Further, what is true for gene arrangements is equally valid for the domains of individual proteins. Functional units serving specific purposes are optimized, and then mixed and shuffled between proteins to serve and adapt to ever-changing cellular demands.

This concept is also true for bacterial signalling transduction-the mechanism by which an organism perceives an input signal and then responds with an appropriate cellular output. Microbes handle high volumes of sensory information with efficiency, specificity, elegance, and modularity. Nearly two decades ago, the concept of communicating modules was beginning to catch fire among microbiologists. Comparative genomics analyses has established that one- and two-component systems are the most widely distributed principles of bacterial signal transduction. Most recently, extracytoplasmic function (ECF) σ factors were added as the third fundamental mechanism of microbial signal transduction.  Link--Relevant Web resources

One-Component Signal Transduction Systems

In the simplest forms of bacterial signal transduction, two functions are fused in a single polypeptide chain. Its sensor domain perceives a mascherfig1signal and then modulates the activity of an effector domain, which orchestrates the cellular response (Fig. 1). Input and output functions can be located in a single domain or can form two separate domains. The umbrella term for the large group of phylogenetically unrelated proteins with these two domains is one-component systems (1CS) (Fig. 1).

An example of 1CS is the lac repressor LacI of Escherichia coli. In response to the input signal of extracellular lactose, this protein releases its inhibiting grip on the lac operator, allowing expression of the lac operon, which encodes functions for using this sugar molecule. LacI, like other bacterial signal transduction effectors, is a transcriptional regulator. In this and other cases, the cellular output consists of differential gene expression by specific genes. Very often, these regulators affect the activity of only a single transcript- here, the lacZYA operon-that encodes proteins devoted to a single purpose-in this case, use of lactose.

Other regulators control a larger number, in some cases even hundreds of target genes, as in global regulators, such as RelA or CRP in E. coli, which adjust the overall gene expression pattern in response to the second messengers ppGpp and cAMP, respectively. While many 1CS function as transcriptional regulators, others regulate a variety of other output domains, including enzymes that make and break cyclic di-GMP, a second messenger that has recently gained a lot of attention.

maschertable1The relatively simple design of 1CS is suited for countless input and output devices, making 1CS the most widely distributed type of bacterial signal transduction system (Table 1). However, combining the input and the output domain on a single protein also has disadvantages, imposing restrictions on signal transduction. Thus, there are very few examples of membrane-anchored, extracellular-sensing 1CS.

Two-Component Signal Transduction Systems

In contrast, many extracellular stimuli that require transmembrane signalling involve the other two categories of bacterial signal transduction, two-component systems (2CS) and extracytoplasmic function (ECF) σ factors (Fig. 1). Remarkably, 1CS and 2CS often share their respective input and output domains, again emphasizing the modularity of bacterial signal transduction. But in contrast to 1CS, these domains are located on two different proteins in the case of 2CS.

A typical 2CS consists of a sensor protein, which functions as a histidine kinase, and an effector protein that is a response regulator. After detecting a specific signal, the histidine kinase autophosphorylates and, in turn, activates its cognate response regulator. This effector protein typically is a transcriptional regulator that modulates expression of its target genes. However, 2CS response regulators have recruited various other output domains, including RNA/protein binding domains or others with enzymatic activitites such as the diguanylate cyclase and phosphodiesterase domains involved in cyclic di-GMP signaling.

Many 2CS use simple and linear pathways from one sensor kinase to one response regulator to control their respective outputs. In contrast to 1CS that predominantly respond to intracellular cues, more than 50% of known sensor kinases respond to extracellular stimuli (Fig. 1). However, the design of 2CS not only simplifies transmembrane signaling, but it also enables many modifications, including amplification and integration of different signals as well as branching of the pathway. The resulting phosphorylation cascades are involved in the control of complex differentiation processes, where many different signals integrate into a single output. Examples include the initiation of endospore formation in Bacillus subtilis and cell cycle control in Caulobacter crescentus.

Thus, 2CS is a very flexible signaling principle based on a highly modular design that is adapted to many cellular needs. Not surprisingly, it is widely distributed in bacteria, archaea, and some lower eukaryotes, with the number of 2CS per genome exceeding 100 in some cases (Table 1).

The concept of 1CS and 2CS as two separate principles of bacterial signal transduction-while intuitive at first glance-remains controversial. If 1CS and 2CS were defined as signaling entities consisting of one or two proteins, respectively, the vast majority of these systems follow this rule. But what if 1CS or 2CS proteins are part of larger regulatory networks and signaling cascades? For example: Is a 1CS protein that participates in the turnover of the cellular net pool of a global second messenger like cyclic di-GMP (i.e., one that contains either a diguanylate cyclase or a phosphodiesterase output domain) really a 1CS? Or do the corresponding proteins together with their substrate represent another fundamental mechanism of bacterial signal transduction, disguised as 1CS or 2CS?

Extracytoplasmic Function (ECF) σ Factors

Alternative σ factors of the extracytoplasmic function (ECF) family are another means by which bacteria direct differential gene expression in response to extracellular cues. In general, σ factors are essential components of RNA polymerase that determine promoter specificity and thereby rates of transcription initiation. In addition to the primary, or housekeeping, σ factors found in all bacteria, most genomes-especially in species with complex life styles-encode alternative σ factors. These proteins redirect RNA polymerase to initiate transcription from alternative promoters after substituting for primary σ factors.

Although the 2CS and ECF σ factors are phylogenetically and biochemically unrelated, they share several mechanistic analogies. For instance, both require two proteins for signal transduction: a typically membrane-anchored sensor protein (histidine kinase or anti-σ factor) and a cytoplasmic transcriptional regulator (response regulator or σ factor, respectively). In both cases the corresponding genes are usually cotranscribed.

However, 2CS and ECF σ factors differ in the way the two proteins communicate with each other and thereby the mechanism of signal transduction. For 2CS, signal transduction is mediated by intramolecular conformational changes, based on transient cycles of phosphorylation and dephosphorylation of both proteins in the presence or absence of a suitable trigger. In contrast, the communication between anti-σ factor and σ-factor is normally based on stable protein-protein interactions in the absence of a stimulus. Thus, the anti- σ factor keeps its partner inactive by titrating it from the pool of freely available σ factors.

maschertable2Once a signal is perceived, the anti-σ factor is inactivated by one of a number of mechanisms (Table 2), thereby releasing the ECF σ factor from anti- σ and activating expression of its target genes. An alternative promoter, which only the corresponding ECF σ factor recognizes, precedes these genes. Because of the nature of this regulation, ECF-dependent signaling always upregulates its target genes, in contrast to 2CS, where the output varies and may entail positive or negative regulation.

Overall knowledge of ECF σ factors is still sparse. However, a recent phylogenetic analysis identified more than 40 distinct subtypes of σ/anti-σ  pairs and demonstrated that this third mechanism of bacterial signal transduction is widely distributed throughout the microbial world (Table 1). While most ECF σ factors have identical do main compositions, the corresponding anti- σ factors embody surprising variety and combinatorial complexity in terms of their conserved modules. A widely distributed and highly diversified antisigma domain (ASD) is present in about one-third of all anti-σ factors associated with ECF-dependent signal transduction. Other anti-σ factors harbor a variety of conserved domains of unknown function. Because of this sequence diversity, it is difficult to recognize anti-σ factors on the basis of sequence analysis.

Further, there is significant—and underappreciated—mechanistic diversity in how anti-σ factors perceive signals and how corresponding σ factors are activated. Nevertheless, potential anti-σ factors from within a conserved subtype of ECF σ factors are usually homologous to each other. This finding suggests that σ-/anti-σ pairs from a given conserved subgroup will have the same mechanism of signal transduction.

Communicating Modules in ECF-Dependent Signal Transduction

mascherfig2Most ECF σ factors interact with membrane-spanning anti-σ factors that harbor a single transmembrane helix (Fig. 2A). In the absence of a stimulus, the anti-σ factor binds the σ factor, rendering it inactive. After detecting a signal, presumably by a surface-exposed protease, the anti-σ factor is degraded in a stepwise manner, eventually freeing the σ factor to interact with RNApolymerase. The two best-understood ECF σ/anti-σ pairs, E. coli RpoE-RseA and B. subtilis σW-RsiW, belong to this group. Here, the cytoplasmic part of the anti-σ  factor harbors an ASD that becomes the interface between anti-σ factor and ECF σ factor and defines partner specificity.

About one-third of known ASD-containing anti-σ  factors coordinate a zinc ion (Fig. 2B). Such a cytoplasmic domain is called a zinc-binding antisigma (ZAS) domain. It plays a crucial role in sensing oxidative stress and mounting an adequate stress response. The two best-understood examples are the SigR-RsrA pair from Streptomyces coelicolor and RpoE-ChrR from Rhodobacter sphaeroides. Again, the σ factor is kept in its inactive state by being bound to its cognate anti-σ factor. Under non-inducing conditions, the zinc cofactor is coordinated by cysteine residues. A change in the cytoplasmic redox potential, indicative of oxidative stress, leads to the formation of a disulfide bond, releasing the zinc cofactor. Thus, the anti-σ factor changes its conformation, releasing its cognate σ factor, which then interacts with RNA polymerase. This mechanism, in contrast to proteolytic degradation of anti-σ factors, is reversible. Hence, the anti- σ factor can be recycled.

Another ECF-dependent signal transduction example is the FecI-FecR pair, which regulates iron acquisition in E. coli and other proteobacteria (Fig. 2E). At first glance, this system contains familiar components, including an anti-σ factor with one transmembrane helix that binds the σ factor FecI via its ASD in the absence of ferric citrate, thereby keeping it inactive. However, σ factor activation is quite different. In this cases, the anti-σ factor remains intact after receiving the signal through protein-protein interactions with a ferric citrate-specific outer membrane porin, FecA. Moreover, FecR is necessary for FecI interactions with RNA polymerase. Thus, it acts as both an anti- σ factor and a mediator of σ factor activity.

Not all ECF σ factors are regulated by anti-σ factors. Sometimes, 2CS components combine with ECF σ factors, forming a variety of signaling pathways across the bacterial kingdom. For instance, the SigE-CseCB module from Streptomyces coelicolor forms a simple yet effective connection between a 2CS and an ECF σ factor (Fig. 2C). CseC is a histidine kinase localized within the cytoplasmic membrane. After envelope stress is perceived, CseC phosphorylates CseB, its cognate response regulator, and it, in turn, induces transcription of sigE, encoding the ECF σ factor. SigE, which lacks a cognate anti-σ  factor, thus enters the cellular σ factor pool and redirects expression to target genes.

The σ-proteobacteria orchestrate their general stress response with an ingenious mechanism that combines 2CS- and ECF-dependent signal transduction (Fig. 2D). In the absence of stress, the σ factor (EcfG) is kept inactive, bound to soluble NepR-like anti-σ factors, which are unlike other anti-σ factors. Once stress is perceived, it is transduced by a 2CS that contains an unusual PhyR-like response regulator that harbors an output domain that is homologous to EcfG-like σ factors. When phosphorylated by its cognate histidine kinase, this protein acts as an anti-anti-σ factor: The anti-σ factor has a higher affinity for phosphorylated PhyR than for its corresponding σ factor. A partner switch releases the σ factor, activating transcription of general stress response genes.

ECF classification efforts identified numerous other alternative modules that could function as input domains in altogether different signaling mechanisms (Fig. 2F). Some novel groups are associated with anti-σ factors that carry conserved domains of unknown function. Other ECF σ factors interact with larger proteins that span the cytoplasmic membrane with six helices, rather than the canonical single helix. Some conserved groups of ECF σ factors have long C-terminal extensions that may play a role in signal transduction, especially since these proteins are often not linked to obvious anti-σ factors. Yet other ECF σ factors are associated with completely unrelated proteins, such as putative serine/threonine kinases or other enzymes. These modules are conserved within individual ECF subgroups, but functional links between these novel protein pairs remain to be discovered.

Where Do We Stand Now-and Where Do We Go from Here?

All the experimentally characterized ECF σ factors are relatively simple signaling units that hardly go beyond single σ/anti-σ pairs mediating direct input- output connections. Presumably this trend, which also fits 2CS, will hold true for the majority of ECF σ factors because the mechanistic features of the modules built into ECF σ/anti-σ pairs restrict the potential of such units. On the other hand, these σ factors offer some options for more complex modes of signal transduction and gene regulation.

Basically, each communicating interface of the cascade can serve as a switchboard to integrate or diversify the signal(s). Two ways to reach higher signaling complexity are based on the σ/anti-σ interaction. Other paralogous anti-σ factors could divide the labor of regulating the cellular pool of a single σ factor. Based on different input signals and binding affinities, subpopulations of the σ  factor could then be gradually released. Likewise, a single anti-σ factor species could inactivate paralogous σ factors, again based on different binding affinities and the strengths of σ/anti-σ  interactions.

The third general mechanism to facilitate a graded response is based on the target promoters. Depending on the degree of sequence conservation within the promoter, high-affinity promoters will be induced at lower σ factor concentrations, while less well-conserved promoters will be activated when higher amounts of σ factor molecules are released. Another way-at least partially-to overcome ECF σ/anti-σ pair signaling limitations occurs when they combine with 2CS proteins. All these mechanisms could combine to form an even more complex stress response network.

ECF σ factors are a widespread and conserved principle of bacterial signal transduction. Moreover, like 1CS and 2CS, ECF σ factors embody a highly modular design, which allows functional domains to shuffle and exchange as separate building blocks across the bacterial kingdom. In the course of evolution, archetypical 1CS, 2CS, and ECF σ/anti-σ pairs succeeded as basic signaling mechanisms in bacteria, presumably because they represent optimized domain combinations. Nevertheless, other combinations persist, as the partner- switching mechanism between 2CS and σ/anti-σ  pairs illustrates. More generally, it seems that bacterial signal transduction is based on a universal pool of numerous signaling domains that perform one of three functions: stimulus perception, intra-/intercomponent communication, or cellular output. In theory, evolution or synthetic biologists may choose such domains at random, as long they are put in a functional order.

SUGGESTED READING

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