Escherichia Coli Multiple [Ni–Fe]

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FEBS Letters 587 (2013) 3562–3566

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Escherichia coli multiple [Ni–Fe]-hydrogenases are sensitive to osmotic stress during glycerol fermentation but at different pHs ⇑ Karen Trchounian a, Armen Trchounian a,b,

a Department of Biophysics, Yerevan State University, 0025 Yerevan, Armenia b Department of Microbiology & Plants and Microbes Biotechnology, Yerevan State University, 0025 Yerevan, Armenia

article info abstract

Article history: Escherichia coli evolves H2 via multiple [Ni–Fe]-hydrogenases (Hyd). This activity under hyper- and Received 7 September 2013 hypo-osmotic stress was investigated with mutants lacking different Hyd enzymes during glycerol Revised 10 September 2013 fermentation. Inhibitory effects of hypo-stress on H2 production was stronger at pH 6.5 in wild type Accepted 11 September 2013 and mutants except fhlA, which encodes a transcriptional activator for Hyd-3, compared with the Available online 20 September 2013 effects of N,N0-dicyclohexylcarbodiimide. These results indicate that Hyd-3 and Hyd-4 are osmosen- Edited by Judit Ovádi sitive at pH 7.5. Hyd-4 and FhlA are implicated in osmotic stress response at pH 6.5. Hyd-1 and FhlA might be osmosensitive at pH 5.5. Thus, osmosensitivity of Hyd enzymes is a novel property that depends on pH. This is signiﬁcant for mechanisms of cell osmoregulation and H production bio- Keywords: 2 Osmotic stress technology when glycerol is used as a fermentation substrate.

1. Introduction Moreover, under anaerobic growth the expression of the hya and hyb operons is different depending on external conditions Escherichia coli and other bacteria are well-known to perform [13–15]. Expression of the hya genes was shown to be induced at mixed acid fermentation of sugars (glucose) evolving hydrogen acidic pH [14] and by the presence of formate [15] whereas the

(H2) as one of the end products [1,2]. Recently, glycerol, like sugars, maximal expression of hyb was at alkaline pH [14] in cultures has been discovered to be utilized anaerobically by E. coli at acidic grown on H2 and fumarate, which was in accordance with pH opti- [3] or alkaline pH [4]. During fermentation of glycerol, besides eth- mum of Hyd-2 [14,16]. Moreover, Hyd-2 activity was observed in a

anol, succinate and the other end products, H2 gas production can more reduced environment [17] and was absent under aerobic be observed. Glycerol fermentation studies are ongoing [4–7] but conditions [18]. Hyd-3 with the formate dehydrogenase (FDH-H) the metabolic pathways and end products, their dependence on forms the formate hydrogen lyase complex (FHL-1) [19], producing

pH and other external factors are still unclear in details. H2 preferably at acidic pH [10,11]. Hyd-4 together with FDH-H is H2 is evolved by E. coli via [Ni–Fe]-hydrogenases (Hyd), which suggested to form the second complex FHL-2 [19], for which a + reversibly oxidize H2 to 2H . The key features of these mem- functional hycB gene product is required at alkaline pH [10]. The brane-bound enzymes are their multiplicity and reversibility fhlA gene encodes a transcriptional activator for the hyc [20] and [1,2]. Hyd-1 (hya) and Hyd-2 (hyb) operate in oxidizing or produc- probably for the hyf [19] operons. It should be noted that some ing mode during glucose or glycerol fermentation, respectively interaction or metabolic cross-talk is suggested for different Hyd

[4,8,9]. Hyd-3 (hyc) and Hyd-4 (hyf) are H2 producing enzymes dur- enzymes [1,9], however, the nature of the link is not clariﬁed. In ing glucose fermentation [9–11] but Hyd-3 can operate in reverse addition, all four Hyd enzymes are proposed to perform H2 cycling mode under certain conditions [12] and during glycerol fermenta- in membrane [2,17,21] but their coordinated operation and role in tion [4,8,9]. cell physiology should be understood.

Previously, it has been shown that H2 production by E. coli Hyd- enzymes during glucose or glycerol fermentation was inhibited by N,N0-dicyclohexylcarbodiimide (DCCD) [8,22], an inhibitor of the ⇑ Corresponding author at: Department of Microbiology & Plants and Microbes F F -ATPase, a key membrane-associated enzyme of bacterial bio- Biotechnology, Yerevan State University, 0025 Yerevan, Armenia. Fax: +374 10 0 1 554641. energetics [24], and disturbed in Datp mutant lacking F0F1 [10,25]. E-mail address: [email protected] (A. Trchounian). Especially, during glucose fermentation at pH 7.5 a link between

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.09.016 K. Trchounian, A. Trchounian / FEBS Letters 587 (2013) 3562–3566 3563

1 Hyd and F0F1 could result from Hyd-4 interaction with F0F1 to sup- NaCl) with glycerol (10 g l ) supplemented at pH 7.5, 6.5 or 5.5 + ply reducing equivalents (H +e ) for energy transfer from F0F1 to for 20–24 h as described elsewhere [4,7–9,22,27]. For some mu- secondary transport [1,24]. This is in agreement with an indepen- tants, where appropriate (see Table 1), overnight growth cultures dent investigation of Barrett’s group [26] revealing a relationship were supplemented with antibiotic kanamycin (25 mg l1). For between FHL and F0F1 during thiosulfate reduction by Salmonella anaerobic conditions, O2 and N2 dissolved in liquid medium were typhimurium. A requirement of F0F1 is also suggested for E. coli bubbled out of the media by autoclaving, after which the vessels H2-oxidizing Hyd-1 and Hyd-2 during glucose and glycerol fer- were closed by plastic press caps. The medium pH was determined mentation [23,27]. by a pH-meter with a selective pH-electrode (ESL-63-07, Gomel

During glucose fermentation H2 production has been deter- State Enterprise of Electrometric Equipment (GSEEE), Gomel, Bela- mined to be sensitive to hyper-osmotic (hyper) and hypo-osmotic rus; or HJ1131B, Hanna Instruments, Portugal) and adjusted using (hypo) stress at neutral and slightly alkaline pH [10,11]. However, 0.1 M NaOH or 0.1 N HCl. Bacteria can grow well during glycerol this effect was abolished when exogenous formate was added, fermentation at different pH [7,8]. which enhanced H2 production by Hyd-3 [11]. This was the ﬁrst indication that osmotic sensitivity of Hyd enzymes and this could 2.2. Preparation of whole cells for assays and osmotic stress be related to their operation mode, and Hyd-4 is suggested to be sensitive to osmotic stress during glucose fermentation. Bacteria grown were washed during 20 min and transferred On the other hand, osmotic regulation by bacteria is an interest- into the assay medium (150 mM Tris-phosphate (appropriate pH) ing but complex phenomenon in cell physiology. E. coli is known to containing 0.4 mM MgSO4, 1 mM NaCl, 1 mM KCl) [4,8–11,22,33]. respond to hyper-stress by changing K+ transport, especially via When cells were washed in distilled water and transferred into the constitutively synthesized TrkA system [28]. Osmotic stress the assay medium bacteria were subjected to a hyper-stress was suggested to lead to conformational changes in TrkA increas- whereas transfer from the other washing solution (0.8 M sucrose) ing its activity. But under sugar fermentation the activity of TrkA into the assay medium was a hypo-stress [29]. This approach is coupled with F0F1 [24]. Probably F0F1 is an osmoregulatory com- was employed to study osmotic stress response by E. coli different ponent of the membrane [30], which in response to hyper stress is mechanisms [10,11,29,30]. In the assays, glycerol was supple- regulating intracellular K+ content by supplying energy from ATP mented at the same concentration as that added to the growth + for K uptake [1,24]. Osmosensitivity of F0F1 can be controlled by medium. For the others, preparation of whole cells for the assays periplasmic proteins: a model on protein functioning as a valve was detailed before [4,8–11,22,33]. is proposed [29]. But many aspects in osmoregulation by F0F1 re- main to be studied. Moreover, the other compatible solutes such 2.3. Redox potential determination and hydrogen production assays as glutamate or proline can be also responsible for osmoregulation under certain conditions [30,31]. The differences in membrane- The redox potential (Eh) in bacterial suspension was determined associated protein content and composition are mediated by os- with a potentiometric method using a pair of redox, titanium–sil- motic stress. Those were determined by proteomic analysis under icate (Ti–Si) (EO-02, GSEEE) and platinum (Pt) (EPB-1, GSEEE; or different conditions [32]. However, osmoregulation in E. coli during PT42BNC, Hanna Instruments, Portugal) electrodes as described glycerol fermentation is not demonstrated and role of Hyd en- previously [4,7,8,10,11,23,33,34]. zymes should be studied. In contrast to Ti–Si-electrode, a Pt-electrode is sensitive to H2 In the present paper E. coli H2-producing activity by Hyd en- and O2 [10,33] and under anaerobic conditions (dissolved O2 was zymes during glycerol fermentation under osmotic stress was bubbled out of the assay media) it can also detect H2 production. investigated. Osmosensitivity of different Hyd enzymes at different Therefore, H2 production rate (VH2) by bacteria was calculated as pHs was determined and a novel role of Hyd enzymes in osmoreg- the difference between the initial decreases in Pt- and Ti–Si-elec- ulation is proposed. trodes readings per time and expressed as mV of Eh per min per mg dry weight of bacteria as described before 2. Materials and methods [4,7,8,10,11,23,33,34]. This electrochemical approach is close to the method with Clark-type electrode employed by different

2.1. Bacterial strains and growth groups [35,36]: a correlation between Eh and H2 production was shown. Moreover, Eh decrease in H2 evolution was determined The E. coli wild type and different mutant strains used in this not to depend on salt content in water solution and the supple- study are listed in Table 1. mentation of H2 itself did not affect external pH [36]. No signiﬁ- Bacteria from an overnight growth culture were transferred and cant difference between Pt- and Ti–Si-electrodes readings was grown under anaerobic conditions at 37 °C in liquid peptone med- detected in bacterial suspension without adding glycerol. More- 1 1 1 1 ium (20 g l peptone, 15 g l K2HPO4, 1.08 g l KH2PO4, 10 g l over, bacterial count alteration in the suspension by approx.

Table 1 Characteristics of E. coli strains used.

Strains Genotype Absent subunit or protein Source and reference

q BW25113 lacl rrnBT14DlacZW116 hsdR514 DaraBADAH33 Drha Wild type, precursor for the other Wood TK. (Texas A&M University, College Station, TX, a BADLD78 mutants USA) [8,12] JW0955 KmRb BW 25113 DhyaB Large subunit of Hyd-1 Wood TK. [8,12] JW2472 KmRb BW25113 DhyfG Large subunit of Hyd-4 Wood TK. [8,12] JW2701 KmRb BW25113 DfhlA FHL activator Wood TK. [8,12] JW2962 KmRb BW 25113 DhybC Large subunit of Hyd-2 Wood TK. [8,12] MW 1000 BW25113 DhyaB DhybC Large subunits of Hyd-1 and Hyd-2 Wood TK. [8,12] SW 1001 KmRb BW 25113 DfhlA DhyfG FHL activator and large subunit of Wood TK. [8] Hyd-4

a Present address: Departments of Chemical Engineering, Biochemistry and Molecular Biology; Pennsylvania State University, University Park, PA, USA. b Resistant to kanamycin. 3564 K. Trchounian, A. Trchounian / FEBS Letters 587 (2013) 3562–3566

8- to 10-fold had no marked effect on Eh value as shown that these enzymes which are situated on the periplasmic side of [8,23,33,34]. the membrane and include Hyd-1 and Hyd-2 [1,2,39] would be-

Using the Durham tube method [10,26],H2 production during come osmosensitive. As mentioned above, mainly Hyd-2 and par- E. coli growth was visualized by the appearance of gas bubbles in tially Hyd-1 are working in H2 producing direction at pH 7.5 the test tubes over the bacterial suspension. This was in good cor- during glycerol fermentation [4,8,37,38] but no effect of osmotic relation with redox measurement of H2 production by bacteria as stress was determined before. This might be explained by Hyd-2 described above. and Hyd-1 changing their conformation or localization within the membrane and, thus, were not directly affected by osmotic stress.

2.4. Others, reagents and data processing This seems likely to be due to a major contribution of Hyd-2 to H2 production during glycerol fermentation resulted from changed Dry weight of bacteria was determined as described previously metabolism and surprisingly inﬂuenced H+ reduction [37]. The [4,8]. For the DCCD inhibition studies, cells were incubated with other possibility is that under osmotic stress metabolic cross-talk the reagent (0.5 mM) for 7–10 min. and interaction between some membrane-associated enzymes

Agar, glycerol, peptone, sucrose, Tris (Carl Roths GmbH, Ger- including Hyd and F0F1 [1,2,9] can be disrupted. This would be with many), DCCD (Sigma, USA) and other reagents of analytical grade Hyd-3 and Hyd-4 since H2 production was markedly decreased un- were used. der hypo-stress at low pH. The effect of pH, which is very complex, Each data point represented is averaged from independent trip- should not be ruled out in this context. licate cultures; the standard errors calculated as described Importantly, the same pH dependent effect was shown for inhi-

[4,8,10,11,23,27,34] are not more than 3% if they are not shown. bition of H2 production with DCCD during glycerol fermentation The validity of differences between average data for each series [4,8,23] therefore comparison with DCCD effects was done with was determined by Student’s criteria (p) as described mutant, which will be described below. [4,8,10,11,23,27,34].

3.2. H2 evolving activity in the mutants with defects in Hyd-1 and 3. Results and discussion Hyd-2 under osmotic stress and effects of DCCD

3.1. H2 evolving activity by E. coli during glycerol fermentation under As it has been shown [4] during glycerol fermentation at pH hyper- and hypo-osmotic stress at different pH 7.5 mainly Hyd-2 and to a lesser extent Hyd-1 are responsible for H2 production by E. coli. However, appropriate mechanisms

Osmotic stress has been shown to affect H2-evolving activity by of these Hyd enzymes and their role in H2 production are not E. coli Hyd enzymes during glucose fermentation at pH 7.5 [10,11]. clear. Moreover, H2 production under certain conditions was

However, during glycerol fermentation at pH 7.5 VH2 by wild type dependent on F0F1 [4,8,23] suggesting a requirement for F0F1. cells was the same under hyper- or hypo-stress (Fig. 1). But at The following findings were important: no Hyd-1 activity band acidic pH, mainly at pH 6.5, VH2 decreased approx. fivefold upon was detected by in-gel activity staining in the atp mutant lacked hypo-stress compared to hyper-stress conditions (see Fig. 1). Thus, F0F1 although H2 production was not inhibited by DCCD in wild osmotic stress effects on H2 evolving activity during glycerol fer- type [22]. mentation was pH dependent. This finding is of interest as mainly Actually, during glycerol fermentation at pH 7.5 E. coli hyaB and

Hyd-2and to a lesser extent Hyd-1 are responsible for H2 evolving hybC single or hyaB hybC double mutants did not show any H2 pro- activity during glycerol fermentation at pH 7.5 [4,8,37,38], whereas duction nor did they show an effect of osmotic stress or DCCD

Hyd-3 or Hyd-4 probably are main H2 producing enzymes [1,2,8]. (Fig. 2). However this was not observed with these mutants at During different stress conditions, also for hypo-stress, sizes or acidic pH (pH 6.5 and 5.5) (Figs. 3 and 4). volume of the periplasmic space of the bacterial cell might be At pH 6.5 DCCD inhibited VH2 in wild type [8]. This was in con- changed: the role of periplasmic proteins could be suggested to trast with that for glucose fermentation under the same conditions regulate membrane-associated proteins conformational structure and shown previously [10,11]. Besides, under hypo-stress at pH 6.5 and activity including F0F1 [29]. Due to this change it is possible VH2 of the wild type was inhibited approx. 4.9 fold, which is twice the level of inhibition with DCCD (Fig. 3). The results conﬁrm an

impact of F0F1 in H2 evolution during glycerol fermentation. This might be due to conformational changes of F0F1 or interaction between different enzymes in the membrane or, it is more probable,

change in proton motive force (Dp) [27]. However, H2 producing Hyd enzymes were also osmosensitive, playing an important role in regulating cell turgor when H+ are transported out of the cells

and H2 cycling across the membrane might be important. This idea seems to suggest that under acid stress Hyd enzymes, mainly Hyd- 1, play role in H+ transport regulation across the membrane for maintaining the intracellular pH suggested by Slonczewski [40].

Interestingly, in hyaB or hybC mutants VH2 was inhibited approx. 2.5 fold by DCCD (Fig. 3). But under hypo-stress it was suppressed strongly approx. 5.7 fold and 6.7 fold, respectively, whereas in hyaB

hybC mutant VH2 was decreased approx. 4.2 (not shown). These data could be explained assuming that deletion of one of the uptake Hyd enzymes is compensated by the other Hyd enzyme but when Hyd-1 and Hyd-2 both are deleted compensatory uptake functions can be changed and maintained by other Hyd enzymes, Fig. 1. H production rates (V )byE. coli BW25113 wild type under hyper- and 2 H2 for example, Hyd-4. Together, these results suggest that under hypo-osmotic stress during glycerol fermentation at different pH. Bacteria were + grown at appropriate pH mentioned; 10 ml/l glycerol was subsequently added into hypo-stress Hyd-1 and Hyd-2 might regulate H pumping through the assay medium at time zero. For the others, see Section 2. the membrane. K. Trchounian, A. Trchounian / FEBS Letters 587 (2013) 3562–3566 3565

3.3. H2 evolving activity in the mutants with defects in Hyd-3 and Hyd-4 under osmotic stress and effects of DCCD

Hyd-3 and Hyd-4 are known as H2 evolving enzymes, they can work in reverse H2 oxidizing mode during glycerol fermentation at pH 7.5 [1,2,4,8]; however this and other properties of these enzymes require clariﬁcation. Interestingly, reversibility has been demonstrated for Hyd-3 during glucose fermentation by Wood’s group [12].

As it was described above DCCD had inhibitory effect on H2 production, indicating involvement of F0F1 in this process. Upon hyper-stress at pH 7.5 in hyfG single mutant VH2 was inhibited by DCCD approx. 6.8 fold but in fhlA single and fhlA hyfG double mu-

tants it was decreased markedly approx. 10 fold (see Fig. 2). VH2 under hypo-stress in fhlA and hyfG mutants was decreased by only

Fig. 2. H2 production rates by E. coli wild type and different mutants with defects in approx. 1.7 fold and threefold, respectively; and in fhlA hyfG mu- Hyd enzymes under hyper- and hypo-osmotic stress at pH 7.5. DCCD (0.5 mM) was tant it was decreased approx. 2.5 fold (see Fig. 2). These data indi- added into the assay medium when indicated. For strains, see Table 1. For the cated that Hyd-3 and Hyd-4 were osmosensitive; alternatively, others, see legends to Fig. 1. FhlA could be involved in osmotic stress effects on H2 production. There was no contradiction with the fact that Hyd-4 might work in + + H2 uptake mode or supply H to Hyd-2: H could be transferred from F0F1 as suggested [4]. Interestingly, osmoregulation of transcription was suggested by change in intracellular solute concentration but under other conditions [41].

Under hypo-stress at pH 6.5, in fhlA mutant VH2 was inhibited approx. 3.8 fold but the inhibition with DCCD was twice more ap-

prox. 7.6 fold (see Fig. 3). This is in agreement with the strong inhibitory effect of DCCD in the same mutant during mixed carbon fermentation [34].InhyfG and fhlA hyfG mutants upon hypo-stress

VH2 was decreased strongly approx. 7.6 fold and 9.1 fold, respectively (see Fig. 3). Interestingly, in these mutants DCCD inhibited

VH2 approx. 2.5 fold and 3.5 fold, respectively. These data suggest that Hyd-4 could be the major Hyd enzyme sensitive to osmotic stress directly.

Under hypo-stress at pH 5.5 in fhlA and hyfG mutants VH2 was decreased approx. 7.6 and 8.5 fold, respectively, and in hyfG fhlA Fig. 3. H2 production rates by E. coli wild type and different mutants with defects in Hyd enzymes under hyper- and hypo-osmotic stress at pH 6.5. For the others, see mutant it was decreased signiﬁcantly – approx. 12 fold (see legends to Fig. 2. Fig. 4). Moreover, DCCD also inhibited VH2 in fhlA single mutant approx. 5.2 fold and in the same manner in hyfG and hyfG fhlA mutants (see Fig. 4). These results suggest that Hyd-3 or FhlA itself as well as Hyd-4 were osmosensitive or play a role in osmoregulation. It could not be ruled out that Hyd-4 has a crucial role in osmotic stress response, which could result in interaction with other

proteins including F0F1 to stabilize the cell turgor and maintain internal pH and Dp. This suggestion is supported by the data above that hyfG and fhlA products are at least osmosensitive and, besides + F0F1, Hyd-1 is also involved in H and H2 cycling to regulate cell turgor.

4. Concluding remarks

E. coli performs glycerol fermentation evolving H2 via multiple and reversible [Ni–Fe]-Hyd enzymes [1,2]. This H2 production has sensitivity to osmotic stress that varies depending on pH. Anal-

Fig. 4. H2 production rates by E. coli wild type and different mutants with defects in ysis of single and double mutants lacking different Hyd enzymes Hyd enzymes under hyper- and hypo-osmotic stress at pH 5.5. For the others, see under hyper- and hypo-stress (see Figs. 2–4) indicates that Hyd- legends to Fig. 2. 3 or Hyd-4 and FhlA protein are osmosensitive at different pH. This might be caused by conformational changes in the Hyd enzymes. Under hypo-stress at pH 5.5 in hybC or hyaB hybC mutant At low pH Hyd-1 and FhlA might be osmosensitive. strains VH2 was decreased approx. 4.9 fold similar to wild type Moreover, at pH 7.5 hyaB and hybC or hyaB hybC mutants dem-

(Fig. 4). But in hyaB single mutant the inhibition was approx. 7.4 onstrated low or residual H2 production as with osmotic stress or fold (see Fig. 2). These data might also indicate that Hyd-1 is in- DCCD. However, this was not observed with these mutants for + volved in H pumping at pH 5.5. Besides, it should be noted that pH 6.5 and pH 5.5 (comp. Figs. 2–4). A requirement of F0F1, which only Hyd-1 dependent Hyd activity has been demonstrated during might play an important role in osmoregulation of bacteria [29], glucose fermentation pH 5.5 [9]. could be also considered. The results may suggest that under 3566 K. Trchounian, A. Trchounian / FEBS Letters 587 (2013) 3562–3566 hypo-stress at low pH Hyd-1 and Hyd-2 might regulate H+ pump- [18] Lukey, M.J., Parkin, A., Roessler, M.M., Murphy, B.J., Harmer, J., Palmer, T., ing through the membrane although different mechanisms are Sargent, F. and Armstrong, F.A. (2010) How Escherichia coli is equipped to oxidize hydrogen under different redox conditions. J. Biol. Chem. 285, 3928– possible. 3938. Thus, the results point out novel properties of Hyd enzymes and [19] Andrews, S.C., Berks, B.C., Mcclay, J., Ambler, A., Quail, M.A., Golby, P. and FhlA protein having pH-dependent sensitivity to osmotic stress. Guest, J.R. (1997) A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-translocating formate hydrogenlyase system. Microbiology 143, 3633– These ﬁndings are important for understanding mechanisms of cell 3647. osmoregulation and for development of H2 production biotechnol- [20] Schlensog, V., Lutz, S. and Bock, A. (1994) Puriﬁcation and DNA-binding ogy when glycerol is used as a fermentation substrate. properties of FhlA, the transcriptional activator of the formate hydrogen lyase system from Escherichia coli. J. Biol. Chem. 269, 19590–19596. [21] Zbell, A.L. and Maier, R.J. (2009) Role of the Hya hydrogenase in recycling of

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