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ScienceDirect Integration of hydrogenase expression and hydrogen sensing in bacterial cell physiology Chris Greening and Gregory M Cook Hydrogenases are ubiquitous in ecosystems and widespread in microorganisms. In bacteria, hydrogen metabolism is a facultative trait that is tightly regulated in response to both external factors (e.g. gas concentrations) and internal factors (e.g. redox state). Here we consider how environmental and pathogenic bacteria regulate [NiFe]-hydrogenases to adapt to chemical changes and meet physiological needs. We introduce this concept by exploring how Ralstonia eutropha switches between heterotrophic and lithotrophic growth modes by sensing hydrogen and electron availability. The regulation and integration of hydrogen metabolism in the virulence of Salmonella enterica and Helicobacter pylori, persistence of mycobacteria and streptomycetes, and differentiation of filamentous cyanobacteria are subsequently discussed. We also consider how these findings are extendable to other systems. Addresses Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand Corresponding authors: Greening, Chris ([email protected]) and Cook, Gregory M ([email protected]) Current Opinion in Microbiology 2014, 18:30–38 This review comes from a themed issue on Cell regulation Edited by Cecı´lia Maria Arraiano and Gregory M Cook For a complete overview see the Issue and the Editorial Available online 5th March 2014 1369-5274/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2014.02.001

Introduction The genomics era has emphasised that metabolism of molecular hydrogen (H2) is widespread among microorganisms and central to all major ecosystems. Selected for a dual role in biological systems, oxidation of this highenergy fuel yields low-potential electrons for respiration and carbon fixation. H2 gas can also be evolved through fermentative and photobiological processes to dissipate excess reductant into the environment [1,2] Excepting obligate hydrogenotrophs (e.g. certain methanogens) [3], most microorganisms restrict H2 metabolism to specific environments and cellular contexts. This is primarily achieved through regulating the transcription of one or more hydrogenases, the metalloenzymes that metabolise H2 [1,2]. Of the three hydrogenase classes, [NiFe]-hydrogenases are the most diverse in structure, function, and distriCurrent Opinion in Microbiology 2014, 18:30–38

bution. They are roughly divided into uptake hydrogenases, which couple the H2 oxidation to the reduction of cellular electron acceptors (e.g. cytochrome b), and evolving hydrogenases, which use reduced cofactors (e.g. NADH) to drive H2 evolution [1]. As recently reviewed [2], the structural, accessory, and maturation genes required for [NiFe]-hydrogenase synthesis are clustered into operons and regulons to enable coordinated expression. Promoters regulating these operons respond to environmental signals and internal cues depending on the physiological roles of the hydrogenase [1,2]. For example, whereas Escherichia coli preferentially grows on organic carbon sources, it upregulates two uptake hydrogenases when these electron donors are scarce [4,5]. The organism is also a facultative fermenter that activates H2 production by a formate hydrogenlyase. To do so, the cell simultaneously senses both the external oxygen concentration ( pO2) and internal redox state [6,7]. Availability of the substrate hydrogen ( pH2) also influences the expression of H2-oxidising enzymes. As a result of fluctuations in the amount of biological H2 production, H2 concentrations vary by approximately five orders of magnitude in ecosystems, from 400 pM at aerobic soil surfaces [8] to 40 mM in anaerobic gastrointestinal tracts [9]. Some proteobacteria that encounter temporal and fluctuations in pH2, for example Ralstonia eutropha in soil and aquatic systems, sense H2 directly and transduce this signal through cascades controlling hydrogenase expression [10]. Other organisms associate H2-producing and H2-oxidising processes; for example, filamentous cyanobacteria (e.g. Nostoc sp. PCC 7120) co-regulate H2-evolving nitrogenases with H2-recycling hydrogenases during cellular differentiation [11]. There are nevertheless organisms that seemingly have no pressure to coordinate hydrogenase regulation with pH2; gastrointestinal proteobacteria (e.g. Salmonella enterica, Helicobacter pylori) are bathed in micromolar concentrations of H2 throughout their lifecycle [9], while certain soil actinobacteria (e.g. Mycobacterium smegmatis, Streptomyces avermitilis) have sufficiently high-affinity enzymes to scavenge even atmospheric H2 [12,13]. Subsequent sections further explore how proteobacterial, cyanobacterial, and actinobacterial systems regulate their hydrogen metabolism to accommodate pH2 and other external and internal cues.

R. eutropha balances heterotrophic and lithotrophic growth modes by responding to pH2 and energy state R. eutropha (formerly Alcaligenes eutrophus) is a strictly respiratory, facultatively aerobic environmental b-proteowww.sciencedirect.com

Regulation of bacterial hydrogen metabolism Greening and Cook 31

bacterium [14]. While a preferential heterotroph, it can also grow mixotrophically and lithoautotrophically using H2 as an electron donor if preferred carbon substrates are absent. Its flexible and efficient growth is one reason for its ubiquity in soil and freshwater and soil biomes. Through studies led by Friedrich, Schwartz, and Lenz, the molecular details of how R. eutropha switches between heterotrophic and lithotrophic growth modes are now well-understood. The organism maintains a core protein inventory for central carbon metabolism in all three growth modes, but upregulates [NiFe]-hydrogenases and Calvin cycle enzymes encoded on the megaplasmid pHG1 during mixotrophic and lithotrophic growth [14,15]. The organism primarily inputs electrons into the respiratory chain using a membrane-bound [NiFe]hydrogenase (MBH) coupled to a b-type cytochrome [16]. It also employs a soluble [NiFe]-hydrogenase (SH) to drive NADH generation through H2 oxidation, thus generating reductant for both CO2 fixation and proton-motive force generation [17]. Though MBH and SH are expressed at low levels constitutively [18], these enzymes can only sustain lithoautotrophic growth when transcribed at high levels from facultative sN-dependent promoters [19]. In contrast to many hydrogenotrophs, these enzymes are highly oxygen-tolerant and are optimally transcribed in aerobic conditions [16,19]. Instead, their expression is regulated according to the availability of electron donors and they are only upregulated if: the preferred organic electron donors are limiting (signal = energy state) and significant quantities of the substrate H2 are present in the environment (signal = pH2) [10,19,20]. Detailed mutational and complementation studies have resolved how R. eutropha senses and responds to H2 concentration (Figure 1). The organism has selected the catalytic centre of a specialised regulatory [NiFe]hydrogenase (HoxB/HoxC) as its means to specifically sense H2; this enzyme shares significant sequence identity with the MBH and has low-level oxidation activity, but cannot sustain lithoautotrophic growth and instead serves a regulatory function [10,21,22]. In the absence of H2 binding, the classical histidine kinase HoxJ exhibits autokinase and phosphotransfer activity; this results in the phosphorylation of the NtrC-type response regulator HoxA, which exceptionally has an inhibitory effect [10]. H2 oxidation at the regulatory hydrogenase leads to the associated histidine kinase being inactivated; it has been speculated that this may depend on electron transfer from HoxB/HoxC to a putative cofactor bound at the PAS domain of HoxJ [22,23]. The Km of the regulatory hydrogenase is estimated to be as high as 25 mM [22]; this ensures that it is only activated when concentrations of H2 are available at levels sufficient for optimal activity of MBH (Km = 6 mM) and SH (Km = 11 mM) activity [24]. HoxA is an NtrC-like response regulator [10] that activates SH and MBH transcription by binding to inverted repeats in their promoters and activating open complex www.sciencedirect.com

formation by the sN-RNA polymerase [25]. The extent of HoxA activation also depends on a signal cascade responding to the available electron donors [19]. While continuous culture studies show that R. eutropha responds to starvation for electrons [20], the molecular details of how this is sensed remains to be understood; the mechanism is apparently distinct from Crp of E. coli and CcpA of Bacillus subtilis [26]. As HoxA is encoded in the MBH operon, its activation results in a positive feedback loop that enables hydrogenase expression to quickly and strongly respond to environmental conditions [18]. Other proteobacteria regulate H2 metabolism through sensing electron sources. H2-sensing systems dependent on regulatory hydrogenases are widely distributed among the a-proteobacteria and b-proteobacteria [1]. They have been characterised to varying extents in Alcaligenes hydrogenophilus [10], the phototroph Rhodobacter capsulatus [27], and the diazotroph Bradyrhizobium japonicum [28]. It is tempting to speculate that other organisms may encode novel H2-sensing systems that remain to be characterised; for example, Geobacter sulfurreducens encodes a [NiFe]hydrogenase operonic with chemotaxis proteins and a fusion protein containing domains homologous to both [FeFe]-hydrogenases and transcription factors [29]. H2 sensing is likely to be especially desirable for organisms in soil and aquatic environments, where H2 concentrations can vary by owners of magnitude spatially and temporally [8]. However, the majority of bacteria either do not need to accommodate pH2 or use other signals to correlate hydrogenase expression with H2 availability. Beyond R. eutropha, carbon catabolite repression is well-understood in E. coli. The membrane-bound uptake hydrogenases Hya and Hyb are not transcribed when preferred organic electron donors are abundant. hyb is repressed in a manner dependent on the cAMP receptor protein (Crp) [26] during growth on sugars [30]. hya is induced when cells enter stationary phase due to carbon-limitation by sS and the transcriptional activator AppY [4,5].

S. enterica facilitates host invasion by differentially regulating uptake hydrogenases with pO2 and redox state The enteric g-proteobacterial pathogen S. enterica employs four [NiFe]-hydrogenases to oxidise and evolve H2 [31]. The formate hydrogenlyase Hyc dissipates the reductant formed during substrate-level phosphorylation as H2 [32]. The three membrane-bound uptake [NiFe]hydrogenases Hya, Hyb, and Hyd can liberate electrons from H2 for their respiratory chain. This allows the organism to harness the micromolar concentrations of H2 fermentatively produced by gut microbiota as a fuel source [9,33]. Despite similarities in structure and function, the three uptake enzymes differ in their redox couplings, oxygen-sensitivity, and regulation. Current models suggest they each contribute to different modes of energy-conservation: Hya primarily recycles H2 proCurrent Opinion in Microbiology 2014, 18:30–38

32 Cell regulation

Figure 1

Low pH2, High Carbon:

HoxC [NiFe]

High pH2, Low Carbon:

RH: Regulatory Hydrogenase

[NiFe]

Catabolite Repressor

HoxB

HoxB

[FeS]

[FeS]

HoxJ Histidine Kinase

His-P

P-Asp

H2

HoxC

?

HoxA

HoxJ

Response Regulator

Histidine Kinase

? Catabolite Repressor

2H+

HoxA Asp

His

Response Regulator

SH PSH

PSH

hoxFUYHWI

Megaplasmid pHG1 PMBH

hoxFUYHWI

Megaplasmid pHG1 PMBH

hoxKGZMLOQRTV

hoxKGZMLOQRTV

MBH

Lithoautotrophic Growth: HoxG [NiFe]

H2 2H

+

Cytochrome bc1 Complex

Cytochrome bo3 Oxidase

H+

H+

HoxK

F1Fo-ATP Synthase

[FeS]

HoxZ Cyt b

UQ / MQ

Cyt c

MBH: Membrane-Bound Uptake Hydrogenase O2

ADP + Pi

H2O

CO2 ↓

↓ ↓

H+

SH: Soluble NADReducing Hydrogenase

RuBP ↓

ATP

HoxI

NAD+ NADH

FMN [FeS] HoxF HoxI

[FeS] HoxU

[NiFe]

[FeS]

HoxH

HoxY

H2 2H+

Biomass Current Opinion in Microbiology

Regulation and integration of lithoautotrophic metabolism in Ralstonia eutropha. Top left: repression of hydrogenases during heterotrophic growth. When pH2 is low and preferred organic electron donors are abundant, hydrogenase transcription is repressed [18]. The histidine kinase HoxJ catalyses autophosphorylation and phosphotransfer to the response regulator HoxA. The phosphorylated form of HoxA is unable to activate transcription [10]. Top right: activation of hydrogenases during lithoautotrophic growth. When pH2 is low and preferred organic electron donors are scarce, the membrane-bound hydrogenase (MBH) and soluble hydrogenase (SH) are transcribed at high levels from sN-dependent promoters. H2 oxidation at Current Opinion in Microbiology 2014, 18:30–38

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Regulation of bacterial hydrogen metabolism Greening and Cook 33

duced during fermentation [32,34]; Hyb sustains anaerobic respiration by coupling H2 oxidation to fumarate reduction [32,35]; and Hyd sustains aerobic respiration by coupling H2 oxidation to oxygen reduction [36]. Hya also contributes to acid resistance [37]. The four hydrogenases of S. enterica are differentially regulated. While multiple factors influence their transcription [31,38], the overriding factor that differentiates them is their expression in aerobic and anaerobic conditions. Employing hydrogenase promoter-lacZ fusions, the Maier laboratory has quantitatively shown that the transcription of the three promoters is finetuned according to the external oxygen concentration and internal redox state. Consistent with their physiological roles, hya, hyb, and hyc are upregulated during anaerobiosis under conditions that promote anaerobic respiration and fermentation [31,32,38]. Unusually, the hyd fusion shows a reciprocal regulation pattern and is most active during aerobic growth; this is a primary reason why Hyd is predicted to not only be oxygen-tolerant, but also oxygendependent [31,36]. Response regulators as FNR, ArcA, and IscR regulate these enzymes as part of global transcriptional responses to aerobic–anaerobic shifts. hya and hyb are under the control of FNR [31], an archetypal transcriptional activator that directly senses oxygen concentration using an O2-labile [4Fe4S] cluster and binds promoters in its intact state [39]. It remains unclear whether FNR has a direct effect on hya and hyb transcription; based on studies on E. coli, it is possible that the regulator instead exerts a coordinated post-translational control on Hya and Hyb by activating the hyp operon encoding their maturases [6]. The redox-sensing twocomponent system ArcBA provides a complementary level of control; activated when the quinone pool becomes more reduced [7], it stimulates hyb transcription yet represses hyd transcription. The redox-active [2Fe2S] protein IscR tightly represses hyb transcription in aerobic conditions [31,40]. Crucially, the findings of the aforementioned pure culture studies are translatable to in vivo settings. Invigorated by their seminal discovery that a hydrogenase is required for H. pylori virulence [9], the Maier group demonstrated that the hydrogenases were collectively essential for virulence of S. enterica serovar Typhimurium in a murine model. While this study suggested the individual enzymes have overlapping or compensatory roles [33], they have subsequently been implicated in specific roles. The Hya mutant has a 20-fold reduction in short-term survival in murine macrophages, possibly due to disruption of pH

homeostasis [34]. Also using a murine model, Hardt’s group recently demonstrated that hyb-mediated consumption of microbiata-produced H2 provides the pathogen with a competitive advantage during the initial stage of infection [41]. In vivo promoter activity assays have demonstrated that all three hydrogenases are significantly and differentially activated compared to medium-only controls when the bacterium is introduced into immune cells [37]. The complex pathogenesis of S. enterica involves invasion of intestinal cells, evasion of the immune system, and dissemination into new tissues; the ability to regulate H2 metabolism to sustain three modes of energy-conservation may contribute to the metabolic flexibility required to fuel these processes and adapt to a range of oxygen tensions [33]. Beyond the enterobacteria, many facultative anaerobic phototrophs and diazotrophs sense oxygen concentration and redox state to regulate hydrogenase synthesis. Classical two-component systems such as RegB-RegA in Rhodobacter capsulatus [42] and RegS-RegR Rhodopseudomonas palustris [43] negatively regulate hydrogenase expression in response to redox state. Likewise, FNRlike proteins commonly activate anaerobic expression of the structural and maturation genes of uptake hydrogenases, including Thiocapsa roseopersicina [44] and Rhizobium leguminosarum [45].

Mycobacteria enhances long-term survival by upregulating hydrogenases during energylimitation and oxygen-limitation Energy-conservation in the slow-growing soil actinobacterium M. smegmatis primarily depends on aerobic respiration of heterotrophic electron donors [46]. However, the Cook laboratory has recently revealed that three differentially regulated, phylogenetically distinct [NiFe]-hydrogenases play a crucial role in the long-term non-replicative persistence of the organism (Figure 2). Continuous and batch culture experiments have both shown that two membraneassociated uptake hydrogenases, Hyd1 and Hyd2, are maximally induced during carbon-limitation [12,47]. In contrast to the micromolar affinity proteobacterial hydrogenases, these nanomolar affinity, picomolar threshold enzymes can scavenge the low concentrations of H2 (0.40 nM) dispersed throughout the Earth’s lower atmosphere. Concurrent with the downregulation of primary dehydrogenases, oxidation of this trace but dependable fuel source may provide sufficient proton-motive force for cells to persist in the absence of carbon sources in ever-fluctuating soil ecosystems. Consistently, the long-

( Figure 1 Legend Continued ) the regulatory [NiFe]-hydrogenase causes inhibition of the associated histidine kinase HoxJ [10,21–23]. The response regulator HoxA is therefore maintained in its active unphosphorylated form and can initiate open complex formation at the sN-dependent promoters PMBH and PSH [10,25]. Bottom: energetics of lithoautotrophic growth. During lithoautotrophic growth, the membrane-bound hydrogenase aerobically generates proton-motive force by transferring electrons from H2 to O2 via several respiratory complexes and electron carriers [16]. The soluble hydrogenase couples H2 oxidation to NAD+ reduction, generating reductant for CO2 fixation through the Calvin cycle [17]. www.sciencedirect.com

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34 Cell regulation

Figure 2

Exponential Growth Hydrogenase Hydrogenase NADH Hyd1 Hyd2 dehydrogenase II

Succinate dehydrogenase

Cytochrome bccaa3 oxidase

F1Fo-ATP synthase

+

H

MQ → MQH2 [FeS]

[FeS] 2Fe2S

2Fe2S

[NiFe]

[NiFe]

H2 2 H+

H2 2 H+

(Tropospheric / Cellular)

+

NADH

+

NAD + H

O2

Succinate Fumarate

H2O ADP + Pi

(Tropospheric)

ATP

+

NAD(P) NAD(P)H

H+

→ → →

CO2 → Organic Carbon→ → → Biomass Carbon Fixation

Glycolysis / TCA Cycle

Carbon Sources

Carbon-Limitation NADH dehydrogenase I

Hydrogenase Hyd1

Cytochrome bccaa3 oxidase

Hydrogenase Hyd2

H+

F1Fo-ATP synthase

H+

MQ → MQH2

+

NADH

NAD + H

[FeS]

[FeS]

[NiFe]

[NiFe]

H2 2 H+

H2 2 H+

+

(Tropospheric / Cellular)

O2

H2O ADP + Pi

(Tropospheric)

ATP +

H

Carbon Sources → → → Biomass + CO2 (Trace) +

NAD(P) NAD(P)H Glycolysis / TCA Cycle

Oxygen-Limitation NADH dehydrogenase II

Hydrogenase Hyd1

Fumarate reductase

Cytochrome Cytochrome bccaa3 oxidase bd oxidase

F1Fo-ATP synthase

H+

MQ → MQH2 [FeS]

NADH

+

NAD + H

+

[NiFe]

Fumarate

Succinate

H2O

O2

H2O

O2 (Trace)

(Trace)

ADP + Pi

H2 2 H+ (Fermentative)

NADPH

Carbon Sources → → → Biomass + CO2

+

NADP +

NAD(P) NAD(P)H Glycolysis / TCA Cycle

FAD [FeS]

2 H+

[FeS]

ATP H+

[NiFe] [FeS]

H2

Hydrogenase Hyd3 Current Opinion in Microbiology

The roles of the [NiFe]-hydrogenases of Mycobacterium smegmatis during different growth conditions. Depicted is a simplified representation of the mycobacterial electron transport chain based on previous transcriptome and physiological studies in M. smegmatis [46,47]. Top: M. smegmatis primarily grows through aerobic respiration of organic electron donors. Co-metabolism of H2 by Hyd1 and Hyd2 maintains an optimal balance between

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Regulation of bacterial hydrogen metabolism Greening and Cook 35

term survival of Dhyd1, Dhyd2, and Dhyd123 strains is compromised during carbon-limitation (C. Greening, PhD thesis, University of Otago, 2013). Microarray and promoter-lacZ fusion studies confirm that expression of the third hydrogenase (Hyd3) is restricted to microaerobic conditions and is controlled by the DosS/DosT-DosR two-component system [12]. In Mycobacterium tuberculosis, the heme-binding histidine kinases DosS and DosT respectively sense redox state and oxygen concentration [48]; both kinases can activate the response regulator DosR, which in turn binds to conserved promoter regions to activate transcription of target genes [49]. To accommodate the accumulation of reduced coenzymes yielded by carbon oxidation, we model that Hyd3 directly couples NADPH oxidation to H2 elimination [12]. This process maintains redox balance in the cell and enhances cell viability 10-fold during adaptation to and survival of oxygen deprivation (C. Greening, PhD thesis, University of Otago, 2013). While H2 metabolism has principally been studied in the context of growth, studies in actinobacteria are increasingly emphasising a role for H2 metabolism in survival. Other actinomycetes, including streptomycetes and rhodococci, also restrict expression of a class of high-affinity [NiFe]-hydrogenases to energy-limitation [13,50]. Constant and Conrad’s fascinating finding that hhyLS mRNA transcripts are confined to spores in Streptomyces sp. PCB7 suggests that H2 scavenging may be central to their persistence [13]. Consistently, these enzymes are also profoundly thermostable and stress-resistant [24]. Though the pathogen M. tuberculosis lacks orthologs of the M. smegmatis hydrogenases, it encodes an Ehr complex [29] similar to the formate hydrogenlyase hyf in E. coli [12]. In support of a possible role in fermentative H2 production, this operon is also under the control of DosR [51]. Intriguingly, a major systems biology study recently modelled that the helix–turn–helix protein Rv0081 cotranscribed with this complex serves as a regulatory hub that interacts with numerous upstream and downstream regulators [52].

Cyanobacteria spatially and temporally restrict hydrogen metabolism from oxygenic photosynthesis Cyanobacteria are a diverse phylum of phototrophs that are widespread in terrestrial and aquatic habitats. The diazotrophic species encode hup hydrogenases to recycle the H2 inevitably produced through the reaction mech-

Figure 3

Vegetative cells:

hupL3′

xisC

hupL5′

9.5 kb

Heterocyst cells: xisC hupL 9.5 kb Current Opinion in Microbiology

Site-specific recombination at hydrogenase loci during heterocyst differentiation in Nostoc sp. PCC 7120. Top: in vegetative cells, the hupL gene encoding the large subunit of a H2-recycling hydrogenase is interrupted by a 9.5 kb element (green box) [11]. Bottom: during heterocyst differentiation, the recombinase XisC is activated and mediates site-specific recombination at the 16-bp repeats (grey triangles) flanking each side of interrupting element [54]. The 9.5 kb element is excised, generating a continuous hupL reading frame. The resultant hup expression enables cells to recover the H2 wastefully produced from the nitrogenase in heterocysts [11].

anism of nitrogenases. Because of the oxygen-sensitivity of the nitrogenase and hydrogenase, these processes are segregated from oxygenic photosynthesis through complex regulatory mechanisms. In filamentous cyanobacteria, oxygenic photosynthesis occurs in undifferentiated vegetative cells, whereas nitrogenase and hup expression is restricted to the terminally differentiated heterocyst cells [11,53]. While studying cellular differentiation of Nostoc sp. PCC 7120, the Golden group serendipitously identified that the organism activated programmed DNA of hydrogenase-coding regions rearrangements (Figure 3). In vegetative cells, the hupL gene encoding the hydrogenase large subunit is interrupted by a 9.5 kb intervening sequence [11]. During differentiation to a heterocyst, this sequence is precisely excised by the sitespecific recombinase xisC to yield a continuous open reading frame [11,54]. Phylogenetic analysis and hybridisation studies suggest that this strategy is employed by approximately half of sequenced filamentous cyanobacteria and is especially common in Nostoc species [55–57]. In contrast, the uptake hydrogenase of Anabaena variabilis is under the control of a global regulator of heterocyst development. qRT-PCR shows that hup expression in

( Figure 2 Legend Continued ) catabolic and anabolic processes, and may provide reductant for CO2 fixation. Middle: during carbon-limitation, there is reduced flux through the electron transport chain due to lack of electron donors. Concurrent with the downregulation of primary dehydrogenases, the upregulated Hyd1 and Hyd2 are proposed to feed electrons into the respiratory chain, with O2 serving as the terminal electron acceptor. This process helps to maintain a proton-motive force and generate ATP at levels that can sustain dormant cells. Bottom: during oxygen-limitation, there is reduced flux through the electron transport chain due to lack of electron acceptors. The cell appears to maintain some electron transport using upregulated terminal oxidases and electrons yielded from the upregulated Hyd1 are a major input. When available electron acceptors are exhausted, substrate-level phosphorylation of organic carbon sources may be responsible for the majority of ATP generation. The DosR-activated Hyd3 dissipates the excess electrons yielded by this process by coupling the oxidation of NADPH to the evolution of H2 [12,47] (C. Greening, PhD thesis, University of Otago, 2013). www.sciencedirect.com

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36 Cell regulation

this organism is controlled by NtcA, likely through direct binding to the promoter region [58]. Current models emphasise that NtrA senses nitrogen starvation by sensing a-ketoglutarate accumulation and initiates a signal cascade leading to activation of the master regulator of heterocyst differentiation HetR [59]. There is also evidence that NtcA directly regulates hup of the unicellular diazotrophic cyanobacterium Gloeothece sp. ATCC 27152 [60]. It is emerging that temporal separation also influences the expression of a second hydrogenase in cyanobacteria. Many unicellular and filamentous species encode a cytosolic [NiFe]-hydrogenase that can reversibly couple the oxidation of NADH to the evolution of H2. It appears to serve as a redox valve that dissipates excess reductant while enabling continued ATP production through photobiological and fermentative processes. In laboratory settings, H2 production by this enzyme is maximal during induction of anaerobiosis and transitions in illumination; this suggests that the enzyme facilitates metabolic switches in cyanobacteria [61]. Consistently, expression of the structural subunits also peaks during the transition from day to night in unicellular cyanobacteria; the expression of hox promoters shows a clear circadian pattern in Synechoccus [62] and Synechocystis [63] species. The wellreported bacteriophytochrome CikA may be the photoreceptor for this process [64]. It is also known that a LexA family protein and an AbrB-like protein specifically bind and activate the hox operons in Synechocystis sp. PCC 6803 [65,66]. However, it is not understood if and how these transcription factors are linked with photoreceptors.

importance of sensing electron availability, redox state, pH2, and pO2 reflects the fundamental roles that hydrogen oxidation and evolution have in biological systems. There is nevertheless considerable diversity in how and why H2 metabolism is regulated in different phyla and ecosystems. Despite decades of investigation, there nevertheless remains an incomplete understanding of the molecular details of hydrogenase regulation in even the more sophisticated models explored in this work, for example, R. eutropha and S. enterica. Further investigation of emerging models, for example M. smegmatis [12], Geobacter sulfurreducens [29], Synechocystis sp. PCC 6803 [63], and H. pylori [9], is also likely to reveal new themes in the regulation and integration of H2 metabolism.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

2. 

Schwartz E, Fritsch J, Friedrich B: H2-metabolizing prokaryotes. In The Prokaryotes: Prokaryotic Physiology and Biochemistry, edn 4. Edited by Rosenberg E, DeLong EF, Stackebrandt E, Loy S, Thompson F.Springer Press; 2013:119-199. This superb book chapter provides a detailed and current insight into the genetics, biochemistry, and physiology of microbial hydrogen metabolism across a wide range of organisms and ecosystems. 3.

Thauer RK, Kaster A-K, Goenrich M, Schick M, Hiromoto T, Shima S: Hydrogenases from methanogenic archaea, nickel, a novel cofactor, and H2 storage. Annu Rev Biochem 2010, 79:507-536.

4.

Atlung T, Kundsen K, Heerfordt L, Brøndsted L: Effects of sS and the transcriptional activator AppY on induction of the Escherichia coli hya and cbdAB-appA operons in response to carbon and phosphate starvation. J Bacteriol 1997, 179:21412146.

Iron and nickel availability controls expression of the H. pylori [NiFe]-hydrogenase It is emerging that metal sensing is central to transcriptional regulation of the gastric e-proteobacterial pathogen H. pylori. During the course of reductive evolution, the 16 Mb genome of this organism has apparently only retained 17 transcription factors [67]. Its transcriptional regulatory network uniquely centres on two pleiotropic metal-responsive regulators, the nickel-sensing NikR and the iron-sensing Fur. Consistent with its metal composition, there is evidence that the maturation and structural subunits of the membrane-bound hydrogenase are also principally regulated by these factors [68,69]. Activity of this enzyme is essential for virulence [9]. Another example where element availability is important is in the archaeon Methanococcus voltae, which represses the synthesis of its [NiFe]-hydrogenases in favour of [NiFeSe]-hydrogenases if selenium is available [70].

Summary Using four primary models, this review focused on how different bacteria regulate and integrate the expression of their [NiFe]-hydrogenases to adapt to ever-changing environmental and cellular pressures. The recurrent Current Opinion in Microbiology 2014, 18:30–38

Vignais PM, Billoud B: Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 2007, 107:4206-4272.

5. 

Pinske C, McDowall JS, Sargent F, Sawers RG: Analysis of hydrogenase 1 levels reveals an intimate link between carbon and hydrogen metabolism in Escherichia coli K-12. Microbiology 2012, 158:856-868. This article provides new insights into the underexplored area of how carbon and hydrogen metabolism are integrated. It suggests that the extent of carbon flow through glycolysis influences the expression of fermentatively expressed uptake hydrogenase hya. It also posits a role for post-transcriptional and post-translational regulation. 6.

Messenger SL, Green J: FNR-mediated regulation of hyp expression in Escherichia coli. FEMS Microbiol Lett 2003, 228:81-86.

7.

Malpica R, Franco B, Rodriguez C, Kown O, Georgellis D: Identification of a quinone-sensitive redox switch in the ArcB sensor kinase. Proc Acad Natl Sci U S A 2004, 101:13318-13323.

8.

Constant P, Poissant L, Villemur R: Tropospheric H2 budget and the response of its soil uptake under the changing environment. Sci Total Environ 2009, 407:1809-1823.

9.

Olson JW, Maier RJ: Molecular hydrogen as an energy source for Helicobacter pylori. Science 2002, 298:1788-1790.

10. Lenz O, Friedrich B: A novel multicomponent regulatory system mediates H2 sensing in Alcaligenes eutrophus. Proc Acad Natl Sci U S A 1998, 95:12474-12479. 11. Carrasco CD, Buettner JA, Golden JW: Programmed DNA rearrangement of a cyanobacterial hupL gene in heterocysts. Proc Acad Natl Sci U S A 1995, 92:791-795. www.sciencedirect.com

Regulation of bacterial hydrogen metabolism Greening and Cook 37

12. Berney M, Greening C, Hards K, Collins DM, Cook GM: Three  different [NiFe]-hydrogenases confer metabolic plasticity in the obligate aerobe Mycobacterium smegmatis. Environ Microbiol 2014, 16:318-330. Using promoter-lacZ fusions, this study demonstrates that the three Mycobacterium smegmatis hydrogenases are maximally expressed during carbon-limitation and oxygen-limitation, and that the global hypoxic response regulator DosR is responsible for activation of hyd3. Combined with phylogenetic and activity studies, these findings are used to model the physiological roles of the enzymes. 13. Constant P, Chowdhury SP, Pratscher J, Conrad R:  Streptomycetes contributing to atmospheric molecular hydrogen soil uptake are widespread and encode a putative high-affinity [NiFe]-hydrogenase. Environ Microbiol 2010, 12:821-829. This study combines gas chromatography with fluorescence in situ hybridisation experiments to show that a specialised high-affinity [NiFe]-hydrogenase is transcribed exclusively in spores of streptomycetes. This supports a role for atmospheric H2 uptake in the long-term persistence of actinobacteria. 14. Pohlmann A, Fricke WF, Reinecke F, Kusian B, Liesegang H, Cramm R, Eitinger T, Ewering C, Potter M, Schwartz E et al.: Genome sequence of the bioplastic-producing ‘‘Knallgas’’ bacterium Ralstonia eutropha H16. Nat Biotechnol 2006, 24:1257-1262. 15. Schwartz E, Voigt B, Zu¨hlke D, Pohlmann A, Lenz O, Albrecht D,  Schwarze A, Kohlmann Y, Krause C, Hecker M, Friedrich B: A proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha H16. Proteomics 2009, 9:51325142. This study compares the proteomes of R. eutropha during lithoautotrophic, mixotrophic, and heterotrophic growth. In support of previous physiological and genomic studies, Schwartz et al. reveal that R. eutropha maintains a central heterotrophic metabolism in all growth modes, while activating hydrogenases and Calvin cycle enzymes when organic electron donors are limiting. 16. Fritsch J, Scheerer P, Frielingsdorf S, Kroschinsky S, Friedrich B,  Lenz O, Spahn CM: The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre. Nature 2011, 479:249-252. Most membrane-bound hydrogenases are inhibited by O2 and are repressed under aerobic conditions. The culmination of numerous studies, this crystal structure explains how Ralstonia eutropha oxidises H2 in aerobic conditions using a specialised oxygen-tolerant hydrogenase containing a unique Cys6[4Fe3S] cluster in the small subunit. 17. Burgdorf T, van der Linden E, Bernhard M, Yin QY, Back JW, Hartog AF, Muijsers AO, de Koster CG, Albracht SPJ, Friedrich B: The soluble NAD+-reducing [NiFe]-hydrogenases from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH. J Bacteriol 2005, 187:3122-3132. 18. Schwartz E, Buhrke T, Gerischer U, Friedrich B: Positive transcriptional feedback controls hydrogenase expression in Alcaligenes eutrophus hydrogenase genes. J Bacteriol 1999, 181:5684-5692. 19. Schwartz E, Gerischer U, Friedrich B: Transcriptional regulation of Alcaligenes eutrophus hydrogenase genes. J Bacteriol 1998, 180:3197-3204. 20. Friedrich CG: Depression of hydrogenase during limitation of electron donors and derepression of ribulosebisphosphate carboxylase during carbon limitation of Alcaligenes eutrophus. J Bacteriol 1982, 149:203-210. 21. Kleihues L, Lenz O, Bernhard M, Buhrke T, Friedrich B: The H2 sensor of Ralstonia eutropha is a member of the subclass of regulatory [NiFe]-hydrogenases. J Bacteriol 2000, 182:2716-2724.

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40. Giel JL, Rodionov D, Liu M, Blattner FR, Kiley PJ: IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli. Mol Microbiol 2006, 60:1058-1075.

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38 Cell regulation

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