The complex regulatory mechanisms of bacteria and archaea help them to survive wide-ranging stresses in varied environments

Joan L. Slonczewski, James A. Coker, and Shiladitya DasSarma

Joan L. Slonczewski is a Professor in the Department of Biology, Kenyon College, Gambier, Ohio, and James A. Coker is a Postdoctoral Fellow and Shiladitya Das- Sarma is a Professor in the Department of Microbiology and Immunology, University of Maryland, Baltimore.

Summary
● Overlapping constitutive mechanisms, operons, and global regulators enable bacteria and archaea to maintain homeostasis.

● Withstanding constant stress or recurring rapid stress may require microorganisms to express specific proteins constitutively.
● When switching between different environmental states, bacteria and archaea typically regulate gene expression via hierarchal regulators such as sigma factors and basal transcription factors.
● Faced with fluctuating stress, bacteria and archaea sometimes manifest distributed independent responses without hierarchal control.


Microorganisms inhabit nearly all environments on Earth, adapting sometimes to a single stressful condition and at other times to coincident stresses. When faced with a single stress, thermophilic and psychrophilic Bacteria and Archaea typically express a fraction of their entire proteome constitutively. In other cases where multiple stresses abound, such as for the opportunistic environmental pathogen Pseudomonas aeruginosa, and for some halophilic archaea (haloarchaea), the microorganisms prove to be extremely versatile, turning on many different sets of genes that permit them to survive a range of different stressors. For example, the haloarchaeon Halobacterium sp. NRC-1 can grow within a salt crystal but also adapts to low-salt environments (see cover photo).

Even those microbes that grow in one relatively constant environment face changes that threaten viability. For example, hyperthermophilic archaea such as Pyrodictium occultum and Pyrococcus horikoshii experience extreme gradients of temperature within shifting hot geothermal and cold ocean currents near thermal vents, inducing either heat- or cold-shock proteins. Similarly, Escherichia coli cells experience rapid shifts in pH when passing through the stomach to the lower gastrointestinal tract, equivalent to 1,000-fold changes in hydrogen ion concentration. Following such rapid shifts, cytoplasmic pH homeostasis of E. coli temporarily fails, but recovers within less than one minute. Such enteric pathogens adapt to multiple environments. For example, E. coli O157:H7 grows and causes disease in humans but can also grow in aquatic systems or within the vasculature of plants. Obligate intracellular pathogens such as Chlamydia species develop spore-like forms to withstand moving from one host to the next.

Our understanding of microbial stress responses is complicated by the fact that organisms experience multiple stresses simultaneously. For example, the haloarchaeon Halorubrum lacusprofundi was isolated from an Antarctic lake that is ice-free due to its high salinity (5 M NaCl). H. lacusprofundi grows for most of the year at subzero temperatures and with minimal dissolved oxygen. The pathogen Salmonella enterica grows within a macrophage phagosome of its mammalian host, where it encounters low pH, oxidative stress, and defensins. In dissecting such responses, our experiments traditionally address one stress at a time, holding other conditions constant. Yet, such one-at-a-time stress tests bear little resemblance to what microorganisms face in their natural environments.

In some cases, microbes appear to anticipate stresses. For example, E. coli cells moving through the gastrointestinal tract respond to increases in temperature as if they were detecting a simultaneous decrease in oxygen. Moreover, starvation induces resistance to oxidative stress, anaerobiosis, and either low or high pH-all conditions that cells might encounter during stationary-phase growth. Similarly, haloarchaeal cells appear to take microaerobic conditions as a signal for desiccation or DNA damage. These observations highlight the fact that we are only beginning to understand how microorganisms integrate responses to diverse stresses.

Here we explore examples of microbial responses drawn primarily from two very different kinds of microorganisms and environments, namely haloarchaea from extremely high-salt lakes and acid-resistant enteric gram-negative bacteria such as E. coli and Salmonella. In both cases, stress integration mechanisms fall into three categories: (1) constitutive expression of responses to a constant or recurring stress, (2) global responses with a hierarchal regulator for switching between different environments, and (3) distributed independent responses to stress fluctuation without hierarchal control.

Constitutive Expression of a Stress Response System

All cells must maintain conditions inside that differ from those outside the cell. Genes that are expressed constitutively and thus work continually to maintain a homeostatic balance inside the cell control many conditions. Some products of constitutively expressed genes help to protect cells from constant or continually recurring stresses. One classic example is flux in salinity or osmotic pressure. To respond to external changes in ionic concentrations, cells from all three branches of life accumulate a variety of small molecules, both ionic and zwitterionic. Multiple potassium and sodium transporters regulate the concentrations of major ionic species. Some of these microbial transporters, such as Kdp and TrkA, include homologs distributed widely across Bacteria and Archaea (Fig. 1 and Fig. 2).

For the haloarchaeon Halobacterium sp. NRC-1, channels and pumps consisting of the proteins KdpABC, NhaC, PchAB, and TrkAH monitor and control potassium and sodium ions. These channels and pumps maintain a high concentration of potassium (about 4.6 M) and sodium ions (1.4 M) inside the cell that is almost the reverse of the extracellular concentrations of these ions. During growth in relatively dilute (2.9 M NaCl) or highly saline (5.0 M NaCl) conditions, transcript levels of nearly all the potassium and sodium channels and pumps do not change. Therefore, the primary response to the constant stress of a changing saline environment occurs without significant changes in expression of these genes, a process which may be too slow for microbes inhabiting hypersaline environments.

In E. coli, the Kdp and TrkA systems take up potassium, maintaining the cytoplasmic concentration at approximately 200 mM; however, this level increases at higher external osmolarities. Osmoregulation in E. coli involves at least 15 transport systems for potassium and small molecules. For example, osmotic upshift activates the high-K_M system Trk, enabling potassium ion uptake in cells at concentrations above 1 mM. Below 1 mM potassium, Trk is progressively supplemented by the low-K_M system Kdp. The Trk system is expressed constitutively, while Kdp expression is modulated in part by osmolarity, via a sensor kinase/response regulator. Neither system is part of a global operon, yet both the constitutive and inducible systems maintain osmotic homeostasis. Furthermore, no one transporter is essential for osmoregulation and each transporter plays other roles besides osmoregulation.

In E. coli, potassium transport also contributes to regulating pH. In the upper colon,pHfluctuates between 6 and 8, challenging the ion transporters and proton pumps in the electron transport system of enteric bacteria with the need to respond rapidly. A strain lacking both Trk and Kdp transport systems requires high extracellular potassium to maintain its cytoplasmic pH below the external pH of 7. Thus, in E. coli the potassium ion concentration contributes both to osmoprotection and pH homeostasis. Yet, potassium transport is only one component among an overlapping set of pH homeostasis systems, some of them constitutive, others under regulons or distributed control (Fig. 2).

Global Expression Responses with Hierarchical Regulators

In many microorganisms, global response regulators may govern large numbers of genes, sometimes following a hierarchal pattern. Some Bacteria and Archaea adapt to dynamic environments by increasing their basal transcription factors, including sigma factors orTATAbinding protein (TBP) and transcription factor B (TFB) (Table 1), to respond more rapidly to changing conditions (Fig. 3 and Table 1).

For members of the domain Archaea, one mode of gene regulation occurs in cells with a small number of basal transcription factors (TFs). In these cells, TBP and TFB bind at all promoters and work in concert with activators/repressors to increase or decrease transcription. For the second mode of regulation, the genome encodes multiple TBP and/or TFBs, and specific TBP-TFB pairings regulate transcription of specific groups of genes, analogous to the sigma factors of bacterial cells or TBP-related factors (TRFs) of eukaryotic cells. Having a larger number of sigma or basal TFs usually correlates with living in a complex habitat. The use of multiple regulators suggests that some microorganisms have expanded their numbers of genes coding these factors, enabling them to respond more effectively to their dynamic environments.

Genomic analysis of Halobacterium sp. NRC-1 uncovered the first example of multiple TBP and TFB genes within an archaeon, and three times as many basal TF genes as any other sequenced nonhaloarchaeon. Discovery of a multiplicity of basal TFs led to the hypothesis that particular TBPs and TFBs form pairs that regulate expression of specific groups of genes. Because only a small number of the basal TFs in Halobacterium are essential, the remaining factors could be responsible for regulating specific groups of genes. Further, certain pairs of nonessential basal TFs regulate substantial fractions, sometimes more than 10% of the NRC-1 genome, including some key genes necessary for responding to heat shock. For example, TbpD and TfbA are nonessential under standard growth conditions. However, their respective knockout strains grow more slowly or fail to survive at elevated temperatures, suggesting that those factors are essential for the heat shock response.

Members of the domain Bacteria have several types of global response regulators that recognize specific groups of promoters, including transcriptional activators and repressors and sigma factors that bind transiently to RNA polymerase. In hierarchal regulons, bacteria also employ activator/repressor systems that work with a sigma factor and RNApolymerase to increase or decrease transcription of specific genes responding to a given stress. In E. coli, sigma factors such as RpoS govern overarching regulons with multiple subregulons that protect against oxidative stress, starvation, and pH extremes. For example, the Gad acid resistance regulon (Fig. 2) encodes duplicate genes gadA and gadB for glutamate decarboxylase, which removes CO₂ from glutamate to yield gamma-aminobutyrate in a reaction that consumes a proton. Meanwhile, the GadC transmembrane antiporter exchanges glutamate for gamma-aminobutyrate, completing a cycle that protects the cell at external pH values that are too low for growth (below pH 4).

Besides the Gad cycle, RpoS regulates other acid-protection mechanisms, including periplasmic chaperones hdeA and hdeB, which remove proteins denatured at low pH, and the cyclopropane fatty acid synthase cfa, which increases the membrane content of cyclopropane fatty acids at low external pH. The RpoS sigma factor, like an archaeal basal TF, offers an effective way to mediate a shift between two different growth conditions, such as growth in an oxygenated oligotrophic pond versus growth in the nutrientrich anoxic colon.

As cells starve in the colon, RpoS activates numerous forms of resistance to what cells may encounter outside the colon, such as extremes of pH and oxidative stress. RpoS-regulated genetic systems are also subject to a number of RpoS-independent mechanisms.

For instance, the Gad regulon includes twocomponent regulators such as EvgAS; AraC-like regulators GadX and GadW; a Lon protease substrate, GadE; small noncoding RNAs, such as GadY; a tRNA-modifying GTPase, TrmE; and PhoPQ-mediated response to metal ions such as magnesium and sodium. Nearly every major type of regulation in E. coli participates in regulating the Gad acid consumption cycle. Each regulator responds to specific environmental conditions encountered with low pH, such as Lon protease termination of the Gad cycle following pH neutralization, or the presence of metal ions through PhoPQ.

In addition to Gad, several other acid resistance mechanisms operate independently of RpoS or any other sigma factor. These distributed independent systems, with their own dedicated regulators, protect against acid depending on other accompanying environmental conditions, such as anaerobiosis and the availability of amino acids.

Distributed Independent Responses to Fluctuating Stress

Some forms of stress induce multiple independent responses with no known hierarchal control. When haloarchaeal cells are exposed to intense solar radiation, high salinity can also reduce the availability of dissolved oxygen, making their habitat microaerobic or anaerobic. To compensate, haloarchaea such as Halobacterium sp. NRC-1 have anti-UV and desiccation protection responses as well as facultative anaerobic capabilities. While NRC-1 generally grows aerobically when degrading organic compounds in its environment, it seems to anticipate oxygen-limiting conditions by expressing metabolic programs that are suited to progressively lower levels of oxygen.

For instance, cells sense microaerobic conditions via PAS-PAC domain-containing regulators, such as Bat and DmsR, which regulate expression of bacteriorhodopsin and the DMSO oxidoreductase complex, respectively. Both regulators activate genes independently, with Bat promoting phototrophic growth, and DmsR promoting microaerobic growth. When conditions become anaerobic, these regulators shut down production of bacteriorhodopsin and DMSO oxidoreductase, and the cells turn on the arginine deiminase pathway via the ArcR regulator. These genetic regulators appear to control independent responses, although the overall effects appear to result from progressively lower oxygen conditions.

Haloarchaeal cells sometimes are exposed to high levels of UV or other radiation, and Halobacterium sp. NRC-1 is almost as radiation resistant as Deinococcus radiodurans. In fact, some Halobacterium mutants are the most radiation-resistant organisms known, with LD50 of 12 KGy. Halobacterium cells have independent adaptations to radiation and desiccation stress, both of which induce double-stranded breaks in DNA.xx These adaptations include carotenoid pigments, which have antioxidant properties, and the capacity to up-regulate production of key DNA repair proteins, including the rfa3 operon, which encodes the Rpa single-stranded DNA binding protein, and radA, which codes for the primary recombination protein in Archaea. Carotenoid pigments such as bacterioruberins and β-carotene in the membrane remove extra electrons and free radicals created by intense solar radiation and UV. Damaged DNA is repaired by a multitude of regulatory systems, including recombinational repair coded by eukaryotic-type repair enzymes. These re sponses act independently, and do not appear to be controlled by a master regulator even as they mitigate the same stress.

When E. coli cells adapt to pH changes, they induce numerous single-operon responses, some of which are independent of known hierarchal regulators, or subject to global regulators unrelated to pH stress. Several pairs of aminoacid decarboxylase and transporter systems are homologous to Gad. These decarboxylase systems include adi (arginine), cad (lysine), and spe (ornithine). In each case, the individual decarboxylase operon includes its own substrate-product antiporter as well as regulators responsive to low pH, anaerobiosis, and the amino acid substrate. The adi operon may be subject to the CysB arginine-scavenging regulator. However, in the case of pH stress, each operon has independent expression regulators, each of which integrates multiple signals.

The four hydrogenase systems, each of which interconverts hydrogen ions and hydrogen gas, form another class that is pH-responsive in E. coli. The hydrogenase operons are induced at low pH under anaerobiosis. How pH controls these reactions is not known. However, hydrogen production predominates at low pH, whereas hydrogen is consumed and protons are released at high pH. In sum, no one "global regulator" is essential for protecting cells when they are exposed to acid. Instead, a broad array of constitutive, sigmacontrolled, and distributed independent mechanisms shares that role.

Conclusions

Maintaining homeostasis in diverse habitats is critical for the survival of many bacterial and archaeal species. Studies of model organisms like E. coli and Halobacterium sp. NRC-1 reveal a variety of regulatory mechanisms that involve several common patterns. In one, constitutively expressed proteins such as the Kdp system respond to constant or recurring rapid stress such as pH homeostasis and osmoprotection.

As part of a second pattern, switching between different environmental states, such as heat or cold, may involve large numbers of genes subject to a global regulator such as a sigma factor or basal transcription factors. But no single condition, such as low pH or high salt, is isolated from other conditions of stress in the environment. Thus, other regulators that depend on multiple stress conditions encountered simultaneously need to regulate any global regulator.

As part of a third pattern, stress responses to multiple conditions can require distinct mechanisms that are independent of any hierarchal control. Examples include non-Gad acid resistance systems, microaerobic or anaerobic stress, and DNA-damaging agents.

ACKNOWLEDGMENTS

Our research is funded by National Science Foundation grant MCB-0644167 to J.L.S. and National Science Foundation grant MCB-0450695, National Aeronautics and Space Administration grant NNX09AC68G, and the Henry M Jackson Foundation grant HU0001-09-1-0002-660883 to S.D.

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