Bacterial approaches to sensing and responding to respiration and respiration metabolites

Abstract

Bacterial respiration of diverse substrates is a primary contributor to the diversity of life. Respiration also drives alterations in the geosphere and tethers ecological nodes together. It provides organisms with a means to dissipate reductants and generate potential energy in the form of an electrochemical gradient. Mechanisms have evolved to sense flux through respiratory pathways and sense the altered concentrations of respiration substrates or byproducts. These genetic regulatory systems promote efficient utilization of respiration substrates, as well as fine tune metabolism to promote cellular fitness and negate the accumulation of toxic byproducts. Many bacteria can respire one or more chemicals, and these regulatory systems promote the prioritization of high energy metabolites. Herein we focus on regulatory paradigms and discuss systems that sense the concentrations of respiration substrates and flux through respiratory pathways. This is a broad field of study, and therefore we focus on key fundamental and recent developments and highlight specific systems that capture the diversity of sensing mechanisms.

Abbreviated Summary

Respiration provides organisms with a means to dissipate reductants and generate potential energy in the form of an electrochemical gradient. Regulatory systems have evolved to promote efficient utilization of respiration substrates, as well as fine tune metabolism to promote cellular fitness and negate the accumulation of toxic byproducts. Herein we discuss regulatory paradigms and systems that sense the concentrations of respiration substrates and flux through respiratory pathways.

I. Introduction

Cellular respiration is the transfer of reducing equivalents from an electron donor via a dehydrogenase enzyme to an electron acceptor using a terminal reductase enzyme. The process is mediated by a series of membrane-associated proteins and typically lipid-soluble quinones that transfer electrons between redox proteins. NADH dehydrogenases often initiate respiration through the transfer of electrons from NADH to quinones. The reduced quinones can then be a source of electrons for the reduction of a terminal electron acceptor (TEA) using a terminal reductase enzyme. Microorganisms can utilize a variety of chemicals, including organic molecules (e.g. succinate, lactate, or formate) and inorganic compounds (e.g. H₂S), as electron donors. Similarly, a diverse array of chemicals can be used as TEAs. The variety of respiratory electron donors and acceptors is a primary contributing factor to the diversity of life and a driving force for transformations of the biosphere and geosphere (Jelen et al., 2016, Falkowski et al., 2008). The energy harnessed from thermodynamically favorable redox reactions can be utilized to build a transmembrane electrochemical gradient composed of ions, which is used to do work including fueling locomotion, transporting molecules against a concentration gradient, and powering ATP synthesis.

Mechanisms have evolved to help microorganisms optimize the use of cellular respiration or fermentation. Cells prioritize the use of electron acceptors that have high redox potentials (E₀’). This is accomplished by using a network of transcriptional regulatory proteins that sense respiration status, the concentrations of electron donors or acceptors, or respiration byproducts and respond by altering transcription of genes that contribute to respiration or related metabolic processes. These sensing mechanisms involve interaction of a sensor protein with one or more ligands. Typically, stimulation of the sensor protein modulates its activity and subsequently alters the affinity of a regulatory protein for specific DNA sequences (operators) located in a target operon promoter. Alterations in the occupancy of the regulator on the activator or operator site modulate positive or negative transcriptional regulation of target genes, respectively. In two-component regulatory (TCR) systems, stimulation of a histidine kinase via association with a ligand or a change in physiological state modulates kinase or phosphatase activity, thereby altering the phosphorylation state of the response regulator (Gao & Stock, 2009). Phosphorylation status of the response regulator governs its affinity for promoter DNA sequences, and ultimately regulates transcription of downstream target genes. In contrast, a one-component regulatory system contains both the input (sensory) domain and output (regulatory) domain in one protein. Altogether, these processes enable bacteria to adapt to changing conditions within the environment and/or shifts in metabolic demands.

II. Transitioning between respiration and fermentation

When the concentrations of respiration electron acceptors decrease, the ratio of reduced to oxidized quinone increases. It also results in an increase in the concentration of electron donors for respiration, including an increase in the NADH:NAD⁺ ratio. Regulatory systems have evolved that respond to variations in the levels of molecules associated with altered flux through the electron transport chains and resulting in adjustments to the expression of genes utilized in redox balance, respiration, and carbon flow. Here we review two sensors (Srr and Arc) that utilizes protein thiols to monitor the redox state of the cellular quinone pool. The two systems are not homologues, but likely have similarities in biochemical mechanism, suggesting that these systems have independently evolved to provide a general means to sense flux through respiratory pathways. We also discuss the transcriptional regulator Rex, which achieves a similar physiological objective, but responds to alterations in the NADH pool, which is utilized as a source of electrons for respiration.

ArcAB

The transition between fermentative and respiratory growth in E. coli is regulated by the ArcAB TCR system (Iuchi & Lin, 1988) (Figure 1A). ArcB is a dimeric transmembrane sensor histidine kinase and ArcA is the cognate cytosolic response regulator. Under anaerobic or microaerobic conditions ArcB undergoes autophosphorylation via intra- and intermolecular phosphotransfer before catalyzing the transphosphorylation of ArcA (Teran-Melo et al., 2018, Iuchi et al., 1990). Stimulation of ArcB is modulated by the redox state of ubiquinone and menaquinone pools (Georgellis et al., 2001, Alvarez et al., 2013). Oxidized ubiquinone, which predominates under aerobic respiratory conditions, inhibits ArcB kinase activity. Conversely, as respiration decreases, the rate of electron flux through the respiratory chain decreases, resulting in an increase in the ratio of reduced to oxidized menaquinone and stimulation of kinase activity (Alvarez et al., 2013).

Figure 1:

(A) A simplified model of ArcAB system function. The ArcB histidine kinase senses the redox status of the membrane-soluble quinone pool through redox-active cysteine residues located in the cytosolic PAS domain. Upon cysteine oxidation and disulfide bond formation, ArcB autophosphorylation activity is low. Reduction of the disulfide bond results in increased ArcB kinase activity, and subsequent phosphorylation of the cytosolic response regulator ArcA. Phosphor-ArcA regulates transcription of downstream target genes via binding to operator DNA and can function as both a transcriptional repressor and activator. (B) X-ray structure of the oxidized SrrB DHp-CA catalytic region (PDB ID 6PAJ). The DHp domain is comprised of helices α1 and α2. The phosphor-accepting histidine is colored in green, and the redox-sensitive cysteine residues (Cys464 and Cys501) are yellow. The two SrrB monomers are each colored purple and red, respectively.

It has been hypothesized that the buildup of oxidized quinones leads to the oxidation of a pair of cytosolic cysteines present in the ArcB PAS domain (Malpica et al., 2004). The cysteines can form a reversible intermolecular disulfide bond resulting in decreased kinase activity (Figure 1A). ArcB can form either one or two intermolecular disulfides, which allow for fine-tuning of kinase activity. ArcB responds to oxidized ubiquinone in vitro; however, an E. coli ubiquinone mutant did not display noticeable alternations in ArcAB-dependent gene expression when cultured under aerobic or anaerobic conditions (Bekker et al., 2010). On the contrary, ArcAB did not respond to anaerobic growth in a menaquinone auxotroph, suggesting that ArcB is surveying the status of the menaquinone pool. The silencing of ArcB kinase activity during the switch to aerobic growth required the presence of oxidized ubiquinone (E’°=100 mV) and the activation of kinase activity during the switch to anaerobiosis required the presence of reduced menaquinone (E’°= −75 mV) (Alvarez et al., 2013). The redox potential of the ArcB cysteine residues was determined to be E’°= −41 mV. These findings have resulted in a model wherein ArcB provides a link between the redox status of the ubiquinone and/or menaquinone pools and the phosphorylation status of ArcA. The regulation of ArcB kinase activity is multifaceted, as it is also stimulated by the fermentation byproducts D-lactate, acetate, and pyruvate (Georgellis et al., 1999, Rodriguez et al., 2004).

Phosphorylated ArcA exists as a homodimer and binds operator DNA in the promoter regions of target genes (Drapal & Sawers, 1995, Lynch & Lin, 1996, Jeon et al., 2001). Chromatin immunoprecipitation studies revealed that the architecture of ArcA binding sites is complex and diverse, likely reflecting regulatory synergy between ArcA and other global transcriptional regulators, such as FNR (Park et al., 2013). Phosphorylated ArcA can function as both a transcriptional repressor and activator; the majority of the Arc regulon consists of genes utilized in the oxidation of non-glycolytic substrates and the adaption to microaerobic and fermentative growth (Lynch & Lin, 1996, Chao et al., 1997, Shen & Gunsalus, 1997, Pellicer et al., 1999b, Pellicer et al., 1999a, Cho et al., 2006, Salmon et al., 2005).

Strains lacking ArcAB are sensitive to reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are byproducts of oxygen (O₂) and nitrate (NO₃⁻) respiration, respectively (Lu et al., 2002, Loui et al., 2009). While it is unknown which ArcAB-regulated factors contribute to these phenotypes, these findings suggest that regulatory systems controlling the expression of respiration components may also regulate factors to mitigate secondary effects of respiration, and thereby promote metabolic robustness.

SrrAB

The SrrAB TCR system is the master regulator aiding the transition between respiratory and fermentative growth in Staphylococcus aureus (Yarwood et al., 2001, Pragman et al., 2004, Pragman et al., 2007). As electron flux through respiratory pathways decreases during the transition from respiratory (O₂ or NO₃⁻) to fermentative growth, SrrAB increases transcription of genes involved in fermentation and decreases transcription of genes encoding TCA cycle enzymes and respiratory components (Wilde et al., 2015). SrrAB also regulates clpC protease expression, and thereby modulates energy homeostasis through managed proteolysis (Mashruwala et al., 2019).

SrrB is a transmembrane histidine kinase consisting of an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region (Figure 1B). Under anaerobic conditions SrrB is stimulated, resulting in the autophosphorylation of a histidine residue within the dimerization and phosphotransfer (DHp) domain and transphosphorylation of the cognate response regulator SrrA. Phosphorylation of SrrA is predicted to induce a conformational change that alters affinity for target DNA. The SrrB ATP-binding catalytic (CA) domain contains a pair of cysteines that stimulate SrrB kinase activity when in their thiol form (Tiwari et al., 2020). Structural and biochemical studies indicate that the cysteines can form an intramolecular disulfide bond, which results in decreased autophosphorylation (Tiwari et al., 2020). SrrB variants lacking the cysteines fail to activate SrrAB-dependent biofilm formation. Strains growing fermentatively or with inactivation of terminal oxidases result in SrrB stimulation, which required the presence of menaquinone (Mashruwala et al., 2017). Collectively, these studies support a model where SrrB is stimulated by the accumulation of reduced menaquinone under non-respiratory conditions via reduction of redox-active cysteine residues within the SrrB CA domain (Kinkel et al., 2013, Mashruwala et al., 2017, Tiwari et al., 2020).

A srrAB mutant is sensitive to both reactive nitrogen and reactive oxygen stressors, suggesting that, like ArcAB, SrrAB controls the expression of factors that metabolize toxic byproducts of respiration. Nitric oxide can inactivate aa₃- and bd-hemoprotein terminal oxidases (Borisov et al., 2004, Vos et al., 2001). Dosing S. aureus with nitric oxide results in SrrAB stimulation, suggesting that SrrB acts as a nitric oxide sensor. In support of this, hmp and scdA, which aid in preventing nitric oxide toxicity, are part of the SrrAB regulon (Mashruwala & Boyd, 2017, Richardson et al., 2006). Inactivation of both nitric oxide synthase (nos) and srrAB confers a reduced aerobic growth phenotype, and SrrAB is a positive regulator of nos expression, suggesting interplay between nos and srrAB (Chaudhari et al., 2017, James et al., 2019). The nitrite generated from nitric oxide decomposition stimulates terminal oxidase activity, and thereby, increases the rate of respiration. Additionally, SrrAB controls the expression of virulence factors, highlighting the importance of responding to respiratory status for pathogenesis (Yarwood et al., 2001, Pragman et al., 2004, Pragman et al., 2007, Wilde et al., 2015).

Rex

NADH generated during catabolism is often oxidized via NADH oxidases that feed electrons into the respiratory chain, decreasing the ratio of NADH:NAD⁺. When respiration is disrupted the ratio of NADH:NAD⁺ increases. If the NADH:NAD⁺ ratio becomes too great, the rate of catabolism will slow because of a lack of NAD⁺.

The Rex transcriptional regulator is widely conserved across gram-positive bacteria where it monitors intracellular redox potential and regulates the expression of genes involved in energy metabolism and the oxidative stress response. The DNA-binding activity of Rex is modulated by the ratio of NADH:NAD⁺ (Brekasis & Paget, 2003). When NADH levels are low, Rex binds to the operators of target genes and represses transcription (Figure 2A). An increase in NADH concentration results in the dissociation of Rex from DNA. The structures of Rex dimers in complex with NADH, NAD⁺, and/or a DNA operator reveal that each Rex subunit contains an N-terminal winged-helix DNA-binding domain and a C-terminal Rossmann fold dimerization domain that binds to NADH/NAD⁺ (Sickmier et al., 2005, Wang et al., 2008, McLaughlin et al., 2010, Wang et al., 2011) (Figure 2B). When bound to NADH, Rex forms a closed conformation in which the DNA-binding domains are tightly packed and occluded from the operator site. Release of NADH enables DNA binding by triggering a change in the Rex dimer conformation. NAD⁺ does not inhibit Rex DNA binding, but instead competes with NADH for binding to Rex, allowing Rex to sense redox poise over a range of NAD⁺/NADH concentrations (Brekasis & Paget, 2003).

Figure 2:

Structure and working model for regulation by Rex. (A) A simplified model for regulation and NADH/NAD⁺ sensing by Rex. During O₂ respiration, when the NADH:NAD⁺ ratio is low, NAD⁺ displaces NADH from Rex, which then binds to the operators of target genes and represses transcription (left). During decreased respiration under microaerobic conditions, an increase in the NADH:NAD⁺ ratio results in binding of NADH to Rex and the dissociation from DNA (right). (B) Left: X-ray structure of the Rex homodimer bound to NAD⁺ and DNA (PDB ID 3IKT). The two Rex monomers are shown in red and grey. The DNA recognition helix (α3) is colored in yellow and bound to operator DNA (purple). One molecule of NAD⁺ (depicted as green spheres) binds to the Rex dimer. Right: X-ray structure of the Rex homodimer bound to NADH (PDB ID 1XCB). Two molecules of NADH, one bound to each monomer, are depicted as blue spheres. Upon NADH binding, the DNA recognition domain is rotated approximately 43° and occludes the recognition helix (yellow) from operator binding.

Rex was first characterized in the actinobacterium Streptoymyces coelicolor, where it represses transcription of the heme biosynthesis operon, the cytochrome bd terminal oxidase operon, and the NADH dehydrogenase system (Brekasis & Paget, 2003). The Rex homolog in Bacillus subtilis (YidH) additionally regulates transcription of lactate dehydrogenase and the formate/nitrite transporter (Schau et al., 2004, Larsson et al., 2005, Gyan et al., 2006). In S. aureus, Rex is a central regulator of NO₃⁻ and nitrite (NO₂⁻) metabolism and fermentation (Pagels et al., 2010). In each of these organisms, conserved Rex binding motifs have been identified in promoter regions upstream of target genes.

III. Prioritizing the usage of O₂ as a terminal electron acceptor

Organisms typically prioritize the use of a terminal electron acceptor based on its redox potential, with molecules possessing higher redox potentials utilized first. This prioritization allows the organism to harvest the maximal amount of energy from respiration redox reactions. Because of the electropositive E₀’ of O₂ (818 mV), its use as a terminal electron acceptor confers energetic benefits and most respiring aerobes have evolved to favor O₂ respiration. Oxygen respiration allows for the complete oxidation of growth substrates yielding more reducing equivalents, which generate greater electrical potential. Oxygen respiration also poses some risks; O₂ and its reduced byproducts can be toxic (Imlay, 2002).

Key to these processes is the ability for bacteria to adapt to O₂ availability, which is genetically achieved by O₂-sensing transcriptional regulators. These proteins typically use one of two prosthetic groups to sense O₂: (i) iron-sulfur (FeS) clusters, which are oxidized by O₂, and (ii) heme, which forms a reversible Fe-O₂ bond. Altering cofactor structure drives conformational changes in the sensor proteins. Below we focus on two transcriptional regulators, FNR and FixLJ, that utilize an FeS cluster and heme, respectively, to directly sense the low levels of O₂ to prioritize usage of cytochrome oxidases with high affinity for O₂, or secondary respiratory metabolites including fumarate (E₀’ 31 mV) and NO₃⁻ (E₀’ 420 mV).

FNR

The fumarate nitrate reductase (FNR) transcriptional regulator is a widely distributed FeS cluster-utilizing O₂- responsive protein (Barth et al., 2018, Lambden & Guest, 1976, Kiley & Beinert, 1998, Crack et al., 2014, Green et al., 2009). FNR is a master regulator of the transition between aerobic and anaerobic growth wherein it controls the transcription of genes involved in anaerobic respiration when O₂ is absent and can function both as a transcriptional activator and repressor (Kang et al., 2005, Overton et al., 2006, Constantinidou et al., 2006, Federowicz et al., 2014, Myers et al., 2013). Under anaerobic conditions, FNR activates expression of genes encoding NO₃⁻, NO₂⁻, and fumarate reductase systems which can be used in the absence of O₂ (Myers et al., 2013, Reents et al., 2006).

FNR proteins typically exist as cytosolic homodimers and have two structural/functional domains: an N-terminal region houses a sensory domain that contains four cysteine residues that act as ligands for the FeS cluster, and a C-terminal domain consists of a DNA-binding helix-turn-helix motif (Figure 3A) (Lazazzera et al., 1996, Volbeda et al., 2015, Mettert & Kiley, 2018). FNR can bind one [4Fe-4S]²⁺ cluster per monomer, which promotes formation of the homodimer by initiating a conformational change that enhances site-specific DNA-binding (Lazazzera et al., 1996, Volbeda et al., 2015, Green et al., 1996, Khoroshilova et al., 1995). Under anoxic conditions, FNR has one [4Fe-4S]²⁺ cluster per monomer and is active in direct gene regulation (Figure 3B). In the presence of O₂, the clusters are rapidly oxidized and converted to [2Fe-2S]²⁺ clusters (Crack et al., 2007, Crack et al., 2004, Khoroshilova et al., 1997). The [2Fe-2S]²⁺-FNR has a decreased affinity for DNA and is inactive in direct regulation. The [2Fe-2S]²⁺ cluster is further degraded upon continued exposure to O₂ and apo-FNR is inactive in DNA binding (Figure 3B) (Sutton et al., 2004). The [4Fe-4S]²⁺ to [2Fe-2S]²⁺ switch in FNR appears to be the major mechanism for O₂ sensing (Mettert & Kiley, 2018, Volbeda et al., 2015).

Figure 3:

Structure and working model for regulation by FNR. (A) X-ray structure of holo-FNR monomer from Aliivibrio fischeri (PDB ID 5E44). FNR typically exists as a homodimer. Each monomer contains a structural and a functional domain. The N-terminal sensory domain consists of three subregions: the β-roll domain (purple), the dimerization α-helix (red), and the FeS binding domain (green). The [4Fe-4S]²⁺ cluster (orange and yellow spheres) is coordinated by four conserved cysteine residues in the FeS binding domain. The C-terminal region of the protein consists of a helix-turn-helix DNA-binding domain (blue). (B) FNR can function as both a transcriptional activator and repressor. Under anoxic conditions, FNR is active in gene regulation and has one [4Fe-4S]²⁺ cluster bound per monomer. [4Fe-4S]²⁺ cluster binding promotes FNR homodimerization and enhances site-specific DNA-binding. In the presence of O₂, the [4Fe-4S]²⁺-FNR is oxidized resulting in rapid conversion to [2Fe-2S]²⁺-FNR. Oxidized FNR is a monomer that has decreased affinity for DNA and is inactive in direct gene regulation. Upon continued exposure to O_2, the [2Fe-2S]²⁺ cluster is further degraded to form apo-FNR.

FixLJ

FixLJ is a heme-based O₂-responsive TCR system that is a regulator of microaerobic respiration and other O₂-sensitive processes in nitrogen-fixing diazotrophic bacteria. The histidine kinase (FixL) is regulated by O₂ binding to a heme iron center within its N-terminal heme-PAS domain. In the absence of O₂, the iron within the heme group assumes a high spin state, promoting autophosphorylation and phosphotransfer to the cognate response regulator FixJ (Gilles-González et al., 1995, Gilles-Gonzalez et al., 1991, Rodgers & Lukat-Rodgers, 2005). Phosphorylated FixJ induces a transcription program via expression of the transcription factors nifA and fixK (Reyrat et al., 1993). FixLJ primarily regulates genes required for adaptation to microxia. The FixLJ-FixK signaling cascade controls transcription of genes encoding a high-affinity cbb₃-type cytochrome oxidase (Nellen-Anthamatten et al., 1998). During low O₂ conditions, some rhizobiales also use FixLJ to regulate NO₃⁻ respiration (Anthamatten & Hennecke, 1991, Mesa et al., 2003, Robles et al., 2006). In select organisms, including the diazotroph Sinorhizobium meliloti and the non-diazotroph Caulobacter crescentus, FixLJ upregulates the single-domain auxiliary regulator FixT, which interacts with FixJ to function as a feedback inhibitor of the FixLJ system (Stein et al., 2020, Garnerone et al., 1999). The C. crescentus FixT binds an O₂-labile FeS cluster (likely a [4Fe-4S] cluster), which stabilizes FixT and prevents degradation by a Lon protease (Stein et al., 2020).

IV. Prioritizing the use of terminal electron acceptors other than O₂

Nitrate sensors (Nar and Nre)

In the absence of the more energetically favorable TEAs (e.g. O₂), several regulatory systems function to detect and respond to the presence of alternate TEAs. Nitrate can function as an electron acceptor for dissimilatory NO₃⁻ reduction (Gonzalez et al., 2006). NO₃⁻ is reduced to NO₂⁻, which can subsequently be reduced to either N₂ or ammonia (Moreno-Vivian et al., 1999). Cells employ transcriptional regulators to sense the presence of NO₃⁻, and often the absence of O₂, to induce the transcription of genes involved in the reduction and transport of NO₃⁻ and NO₂⁻. These transcriptional regulators include the NO₃⁻ and/or O₂ sensing TCR systems Nar and Nre.

NO₃⁻- and NO₂⁻-dependent gene expression in E. coli and other proteobacteria is regulated by the Nar TCR system. While most TCR systems consist of one sensor histidine kinase and one response regulator, the E. coli Nar system atypically consists of two TCR systems (NarXL and NarPQ) that independently detect NO₃⁻ and NO₂⁻ to control transcription of genes involved in anaerobic respiration and fermentation (Rabin & Stewart, 1993). These TCR systems are paralogs that diverged during genome duplication (Stewart, 2003). The kinase activity of NarQ is stimulated to similar levels by NO₃⁻ or NO₂⁻, while the NarX is stimulated by NO₃⁻ (Lee et al., 1999, Williams & Stewart, 1997). While NarQ exhibits similar phosphotransferase activity for both NarP and NarL, NarX preferentially phosphorylates NarL (Noriega et al., 2010).

The Nar system controls transcription of genes involved in anaerobic respiration and fermentation. Nar activates expression of genes encoding NO₃⁻ reductase and represses transcription of fumarate reductase and DMSO/TMAO reductase genes (Darwin et al., 1997). DNA footprinting experiments have also demonstrated that Nar interacts with regulatory sequences upstream of the genes encoding formate dehydrogenase, a NO₃⁻ exporter, and pyruvate-formate lyase (Li et al., 1994, Walker & DeMoss, 1994, Kaiser & Sawers, 1995).

In the Staphylococci, the O₂ and NO₃⁻ sensing NreABC controls the expression of genes involved in dissimilatory NO₃⁻ and NO₂⁻ reduction, and the subsequent metabolism of nitrogen-containing metabolites and fermentation (Fedtke et al., 2002, Schlag et al., 2008). The system consists of the cytosolic sensor histidine kinase NreB, the cognate response regulator NreC, and the GAF-domain containing NO₃⁻ sensor NreA (Fedtke et al., 2002, Kamps et al., 2004). NreB is a O₂ sensor that utilizes an FNR-type labile [4Fe-4S]²⁺ cluster (Kamps et al., 2004, Müllner et al., 2008). Under anoxic conditions, [4Fe-4S]²⁺-NreB has kinase activity and phosphorylates NreC, altering its affinity for operator DNA (Kamps et al., 2004, Müllner et al., 2008) (Figure 4). Phosphorylation of NreC is reversable due to endogenous phosphatase activity of NreB. The phosphatase activity of NreB is modulated via the NO₃⁻ sensor NreA (Klein et al., 2020, Unden & Klein, 2021). Apo-NreA inhibits NreB phosphatase activity and inhibits NreC phosphorylation (Nilkens et al., 2014, Niemann et al., 2014, Klein et al., 2020). When bound to NO₃⁻, NreA shows decreased binding to NreB, resulting in stabilization of phosphorylated NreC.

Figure 4:

Structure and working model for regulation by NreABC. (A) Structure of NreA of Staphylococcus carnosus with bound NO₃⁻ (PDB ID 4IUK). NreA consists of a single GAF domain (green chain). Bound NO₃⁻ is depicted as blue and red spheres. The dipole moment of helices α3 and α6 (yellow) stabilize the negative charge of the NO₃⁻ anion. (B) Nre is a O₂- and NO₃-sensing regulatory system. The NreB histidine kinase utilizes a [4Fe-4S]²⁺ cluster for O₂ sensing. Under anoxic conditions, [4Fe-4S]²⁺ NreB has kinase activity and phosphorylates the NreC response regulator (center panel). NreC regulates the transcription of target genes, which are involved in NO₃⁻ and NO₂⁻ reduction and metabolism. In the presence of O₂, the [4Fe-4S]²⁺ cluster of NreB is rapidly oxidized (left panel). NreB has lower kinase activity in the [2Fe-2S]²⁺ and apo forms. In the presence of NO₃⁻, transcription of Nre target genes is hyperactivated via the NO₃⁻ sensor NreA (right panel). NreA inhibits NreB kinase activity, which is relieved upon NO₃⁻ binding. In the absence of nitrate, NreA converts NreB from the kinase to the phosphatase state, leading to the dephosphorylation of NreC (center panel).

Sensors of arsenic and trimethylamine N-oxide (ArsR and TorSR)

In some environments, bacteria generate energy by respiring arsenate (As(V)), reducing it to arsenite (As(III)). Arsenate respiration is regulated by the homodimeric arsenite-responsive transcriptional repressor ArsR. Upon entering the cytoplasm, As(III) binds to a cysteine triad site in ArsR, which induces a conformational change and dissociation from operator DNA (Prabaharan et al., 2019). Each ArsR monomer contains an As(III) binding site (Figure 5A). It is not clear whether As(III) binding to both sites is necessary to produce conformational changes and derepression, and/or if As(III) binding is cooperative. Recently, an ArsR from Shewanella putrfaciens was described that is selective for methylarsenite (MAs(III)) (Chen et al., 2017).

Figure 5:

Structure and working model for regulation by ArsR. (A) Structure of As(III)-bound ArsR homodimer from Corynebacterium glutamicum (PDB ID 6J0E). ArsR regulators are homodimers. Each ArsR monomer (blue and gray chains) binds one As(III) ion (purple spheres). The As(III) binding site is coordinated by three conserved cysteine residues (yellow sticks). The ArsR dimer interface is formed by α-helices 1 and 5 (green), and DNA binding is mediated via α-helix 4 (red). (B) Model of ArsR regulation in Escherichia coli plasmid R773. Arsenic enters the cells as either As(III), which is taken up by the aquaglyceroporin GlpF, or As(V), which is taken up by Pst and Pho phosphate permeases. ArsR is an As(III)-responsive transcriptional repressor that regulates transcription of the arsRDABC operon. In the absence of As(III), ArsR binds to the ars promoter to prevent transcription. In the presence of As(III), ArsR dissociates from the DNA, allowing the expression of target genes: ArsAB is an As(III) efflux pump, ArsC is a cytosolic reductase that reduces As(V) to As(III), and ArsD is an As(III) metallochaperone that transfers As(III) to ArsAB. It has also been demonstrated that ArsD has weak transcriptional repressor activity.

ArsR directly regulates the arr operon, which encodes a periplasmic As(V) reductase ArrAB (Saltikov & Newman, 2003, Murphy & Saltikov, 2009). FNR also has a role in arr regulation in Shewanella species, suggesting an interplay between O₂ sensing and As(V) respiration, in which respiration of O₂ is prioritized over arsenate as an electron acceptor (Murphy et al., 2009). Specifically, arr is activated by FNR under anaerobic conditions (Murphy & Saltikov, 2009). During arsenic exposure, bacterial cells require different set points to regulate the synthesis of As-respiring and As-detoxifying genes. In some bacteria, including Shewanella, ArsR also directly regulates the ars arsenic detoxification operon. In these organisms, ArsR co-regulates both arsenate respiration in the periplasm and As(III) detoxification in the cytosol (Murphy & Saltikov, 2009). Work by Saltikov et al. found that 1000-fold less As(III) was required to activate transcription of the arr operon than the ars operon (Saltikov et al., 2005). This suggests that the promoters may have slightly different operators with different affinities to holo-ArsR, or that additional regulatory elements may affect transcription. In support of the latter, studies using the E. coli plasmid R773 found that the ars operon encodes for two trans-acting transcriptional repressors: ArsR and ArsD (Chen & Rosen, 1997). The primary gene targets of these regulators is the arsRDABC operon, which encodes a cytosolic As(V) reductase (ArsC) and an As(III) efflux transport system (ArsAB) (Wu & Rosen, 1993, San Francisco et al., 1990) (Figure 5B). ArsD is a weak transcriptional repressor that has a 100-fold lower affinity for As(III) than ArsR and requires 10-fold more As(III) than ArsR to derepress ars transcription. More recently, it was demonstrated that ArsD also functions as a metallochaperone that transfers As(III) to the ArsAB efflux system and stimulates ArsA ATPase activity at low As(III) concentrations (Lin et al., 2006, Ajees et al., 2011, Yang et al., 2010).

Under anaerobic conditions, some bacteria can utilize trimethylamine N-oxide (TMAO) for respiration via its reduction to trimethylamine. In E. coli, TMAO-based respiration is regulated by the TorSR TCR system (Simon et al., 1994, Jourlin et al., 1996). TorSR is comprised of the transmembrane sensor histidine kinase TorS and the OmpR-family response regulator TorR. TMAO activates TorS kinase activity and a downstream phosphotransfer cascade, leading to formation of phosphorylated TorR (Jourlin et al., 1997). In the absence of TMAO, TorS phosphatase activity reverses this phosphorelay (Ansaldi et al., 2001). Importantly, TorS does not sense TMAO directly. Rather, the TorSR system is stimulated by TMAO binding to the periplasmic binding protein TorT, which forms a complex with the periplasmic detector region of TorS (Baraquet et al., 2006). TMAO binding to TorT is thought to promote a series of conformational changes within the TorS-TorT complex, which leads to TorS activation.

In the presence of TMAO, phosphorylated TorR activates expression of TMAO respiration machinery (torCAD) via binding to four cis-acting direct repeats upstream of the operon (Simon et al., 1995, Ansaldi et al., 2000). The torC gene encodes the membrane-anchored cytochrome component of the TMAO reductase. The periplasmic TorA protein is the terminal reductase that receives electrons from TorC, and TorD is a cytoplasmic protein that acts a chaperone for the maturation of TorA (Gon et al., 2001a). Interestingly, TorC also plays a role in regulating TorSR activity; in the absence of its required heme cofactors, apo-TorC interacts with TorS and prevents phosphorylation of TorR (Ansaldi et al., 1999, Gon et al., 2001b). The torCAD machinery is believed to be the only regulatory target of TorR (Shimada et al., 2018). A recent study revealed that the responsiveness of E. coli to TMAO is finely tuned to the relative abundances of TorT and TorS; an excess ratio of TorT to TorS fully activates torCAD transcription, whereas an excess of TorS leads to weak response even in the presence of TMAO (Roggiani & Goulian, 2015).

IV. Integration of signals for a tailored transcriptional response

A multitude of respiratory regulatory systems work in conjunction with one another to control gene transcription and achieve maximal energy generation, redox homeostasis, and metabolite detoxification. The integration of respiratory signals is key for a tailored transcriptional response upon environmental changes, thereby ensuring optimal fitness.

The narGHJI operon encodes the respiratory NO₃⁻ reductase in E. coli. Transcription of this operon is induced by holo-FNR in the absence of O₂ (Chippaux et al., 1981, Myers et al., 2013). In the presence of NO₃⁻, phosphorylated NarL binds upstream of the FNR binding location and increases narGHJI transcription (Li & DeMoss, 1988). FNR is required for transcriptional activation, resulting in a “two-step positive control” in which the absence of O₂ is dominant determinant (Stewart, 1982). This two-step regulation is biochemically distinct, but functionally similar, to the mechanism of NreABC regulation of transcription of NO₃⁻ reductase expression in Staphylococcus aureus discussed above (Unden & Klein, 2021) (Figure 2).

The fumarate reductase enzyme complex, encoded by the frd operon, allows E. coli to utilize fumarate (E₀’ 31 mV) as a TEA for respiration. Regulation of frd occurs in response to the levels of O₂, NO₃⁻, and fumarate (Jones & Gunsalus, 1985). In the presence of fumarate, the DcuSR TCRS increases levels of phosphorylated DcuR, which can serve as a transcriptional activator of frd (Janausch et al., 2004, Abo-Amer et al., 2004). Under anaerobic conditions, FNR activates expression of frd (Jones & Gunsalus, 1987). In the presence of NO₃⁻, frd expression is repressed by the Nar system (Kalman & Gunsalus, 1989). Therefore, transcription of frd only occurs in the absence of O₂ (E₀’ 818 mV) and NO₃⁻ (E₀’ 420 mV). This co-regulation of frd highlights the hierarchical preference of respiration substrates (O₂ > NO₃⁻ > fumarate), where control of electron flow is coordinated to prioritize molecules with higher E₀’ (Gunsalus, 1992). It also ensures that fumarate reductase, which can donate an electrons to O₂ generating superoxide, is not expressed in the presence of O₂ (Imlay, 1995).

The thermodynamic output from TMAO reduction is less than O₂. However, counterintuitively, TMAO can be used as a respiratory electron acceptor aerobically in E. coli (Ansaldi et al., 2007). This behavior contrasts from the utilization of alternate TEAs in E. coli. It has been shown that cell-to-cell variability in strict dependence upon O₂ respiration benefits the population (Roggiani & Goulian, 2015). This is thought to occur through a bet-hedging strategy that ensures E. coli metabolism is primed to use TMAO as an electron acceptor when there is a sudden, drastic drop in O₂ tension. Moreover, IscR is a transcriptional regulator that utilizes a O₂ labile FeS cluster (Schwartz et al., 2001). Apo-IscR represses transcription of torS and torT during aerobic growth resulting in “noisy” transcription of torCAD. This repression is relieved by holo-IscR in the absence of O₂. Interestingly, a temperate phage has been identified that can integrate between torS and torT, resulting in increased torS transcription (Carey et al., 2019). Increasing the ratio of TorS to TorT decreases transcription of torCAD. Therefore, the presence of the prophage decreases the ability of E. coli to prime metabolism for the use of TMAO upon a rapid decrease in O₂ tension.

V. Summary and future directions

Determining the direct stimuli and mechanisms of signal transduction for regulatory systems that sense and respond to respiration is necessary for understanding regulator function. Some of these regulatory systems control the expression of virulence factors that aid pathogenesis (i.e. SrrAB). Uncovering their stimuli will provide clues as to the host-derived molecules that inhibit respiration and will help gauge the importance of respiration for pathogen fitness in vivo. Modulating the activities of these regulatory systems using small molecule chemotherapeutics could provide a means to disarm pathogens without imparting the selective pressure of bacteriostasis or death. This strategy could decrease the likelihood of developing resistance to the chemotherapeutic and the adverse side effects associated with altering the native microbial populations (Johnson & Abramovitch, 2017). Recent work has also focused on an increased understanding of how respiration shapes microbial communities, especially in the context of gastrointestinal microbiota (Stacy et al., 2021, Winter et al., 2013, Byndloss et al., 2017).

The diversity of regulatory systems that respond to respiration or respiration-associated signals and their co-regulation of genes associated with fermentation and/or respiration highlights the importance of accurately modulating respiration and prioritizing the use of the respiration substrate that best promotes fitness. Many bacteria contain more than one respiration regulator, which aids the integration of respiration associated signals into the physiological response. Determining the overlap of regulons and the speed of regulator response will provide information about how respiration is tethered to the metabolic network and whether energetics drives the utilization of specific respiration substrates. It is likely that evolution in specific niches, and/or the toxicity of respiration substrates/products, also influences the ordered utilization of respiratory substrates.

Genome-wide analyses will help define the direct and indirect regulons of respiratory regulatory systems. While elegant studies have been conducted in E. coli, most of the regulatory systems discussed still necessitate rigorous inquiry, especially in other organisms. Such studies will aid in gene classification and in assigning biochemical functions to open reading frames, thereby aiding genome annotation. These regulons and annotations are necessary to build interpretable models of gene regulation that will help predict phenotypes from genotypes.