Escherichia coli is able to ferment glycerol and produce H2 by different Hyds (hydrogenases). Wild-type whole cells were shown to extrude H+ through the F1Fo-ATPase and by other means with a lower rate compared with that under glucose fermentation. At pH 7.5, H+ efflux was stimulated in fhlA mutant (with defective transcriptional activator of Hyd-3 or Hyd-4) and was lowered in hyaB or hybC mutants (with defective Hyd-1 or Hyd-2) and hyaB hybC double mutant; DCCD (dicyclohexylcarbodi-imide)-sensitive H+ efflux was observed. At pH 5.5, H+ efflux in wild-type was lower compared with that at pH 7.5; it was increased in fhlA mutant and absent in hyaB hybC mutant. Membrane vesicle ATPase activity was lower in wild-type glycerol-fermented cells at pH 7.5 compared with that in glucose-fermented cells; 100 mM K+ did not stimulate ATPase activity. The latter at pH 7.5, compared with that in wild–type, was lower in hyaB and less in hybC mutants, stimulated in the hyaB hybC mutant and suppressed in the fhlA mutant; DCCD inhibited ATPase activity. At pH 5.5, the ATPase activities of hyaB and hybC mutants had similar values and were higher compared with that in wild-type; ATPase activity was suppressed in hyaB hybC and fhlA mutants. The results indicate that during glycerol fermentation, H+ was expelled also via F1Fo. At pH 7.5 Hyd-1 and Hyd-2 but not FhlA or Hyd-4 might be related to F1Fo or have their own H+-translocating ability. At pH 5.5, both Hyd-1 and Hyd-2 more than F1Fo might be involved in H+ efflux.

INTRODUCTION

Escherichia coli is known to demonstrate a mixed-acid fermentation of sugars (glucose) or glycerol under anaerobic conditions and this results in the formation of lactic acid, formic acid and other organic acids and alcohols such as ethanol, and also it forms carbon dioxide (CO2) and releases molecular hydrogen (H2) [1,2]. Glycerol metabolism in E. coli was thought to be restricted to respiratory conditions, but only recently it was reported that E. coli can ferment glycerol under anaerobic conditions [2]. It represents a relatively simple cluster of biochemical reactions leading to 3-phosphoglyceraldehyde, the entry point to the lower steps of glycolysis [3,4]. Availability, low prices and high degree of reduction make glycerol an ideal feedstock to produce reduced chemicals and fuels via anaerobic fermentation [5].

It was shown that, during fermentation of glucose and glycerol, the membrane-associated enzyme FHL (formate hydrogen lyase in Escherichia coli) catalyses formic acid oxidation to CO2 and H2 [2,6]. FHL activity has been found in various bacteria inhabiting different biotopes, under aerobic and anaerobic conditions [79]. Bacteria living under anaerobic conditions accumulate energy during fermentation and use H2 production to utilize excess reducing equivalents (H++e), for which formic acid could be the main sources.

In E. coli, FHL is a complex that consists of two enzymes: Fdh-H (formate dehydrogenase H) and Hyd (hydrogenase). Formic acid acts as the actual electron donor for FHL, whereas H+ is the only terminal acceptor. This means that both components of FHL, namely Fdh-H and Hyd, should contain oxidation–reduction centres, and electron transfer from Fdh-H to Hyd requires some carriers that might be associated with Hyd subunits.

E. coli possesses four membrane-bound Hyd enzymes [1012]: two of the enzymes, Hyd-3 (encoded by the hyc operon) and Hyd-4 (encoded by the hyf operon), are responsible for H2 production, and the other two enzymes, Hyd-1 (encoded by the hya operon) and Hyd-2 (encoded by the hyb operon), are responsible for H2 uptake and oxidation under glucose fermentation. However, Wood and co-authors recently obtained results suggesting that Hyd-3 can also operate in the reverse direction and has significant H2 uptake activity like Hyd-1 and Hyd-2 [13]. No reversible mode of functioning is suggested for the other Hyd enzymes, although lately Trchounian and Trchounian [14] have shown that Hyd-2 is able to function in reversible, H2 formation mode upon fermentation of glycerol.

It was demonstrated that, during glucose fermentation depending on growth pH, E. coli FHL exists in two forms, which differ from each other mainly by the presence of Hyd-3 (at acidic pH) and Hyd-4 (at neutral and slightly alkaline pH) respectively [11,12,15]. Moreover, Hyd-3 and Hyd-4 might have H+-translocating activity, suggesting a role for them in intracellular pH regulation [12,16]. Interestingly, H2 production by FHL in E. coli fermenting glucose [17,18] or glycerol [14] at neutral and slightly alkaline pH has been shown to be sensitive to DCCD (dicyclohexylcarbodi-imide). It should be noted that DCCD is a non-specific inhibitor of the F1Fo-ATPase; it can also inhibit other membrane mechanisms involved in H+ translocation [19,20]. However, DCCD-resistant H+ efflux under point mutation in the atpB gene leading to changes in F1Fo functioning [21] was shown with E. coli grown under aerobic conditions [22] or under anaerobic conditions but in the presence of NO3 [23], pointing out the selective inhibition of E. coli F1Fo by DCCD.

F1Fo catalyses the terminal step in oxidative phosphorylation utilizing the ΔμH+ (protonmotive force) to drive ATP synthesis [24]. Under fermentation conditions, F1Fo becomes an ATP-driven H+ pump to generate ΔμH+. Under these conditions, E. coli F1Fo has direct involvement in secondary solute transport systems such as TrkA (the constitutive low affinity K+ uptake system in E. coli) [25,26]. Energy of ATP is transferred from F1Fo to TrkA through a dithiol–disulfide interchange; reducing equivalents are required for the energy transfer; those might be supplied through F1Fo and Hyd-4 at neutral and slightly alkaline pH. Thus it was proposed that F1Fo is required for enzymes of anaerobic oxidation–reduction such as FHL in E. coli [10,17,27] and Salmonella typhimurium [8].

In accordance with the data, recently obtained by Trchounian and Trchounian [14] in our laboratory, that Hyd-2 rather than Hyd-1 is responsible for H2 formation which is sensitive to F1Fo inhibitors under glycerol fermentation, in the present study we have examined the H+ efflux and ATPase activity of the glycerol-fermented E. coli wild-type and mutants of different Hyd enzymes in slightly alkaline and acidic media. DCCD effects on both H+ efflux and ATPase activity were investigated. It was shown that, at pH 7.5 and pH 5.5, E. coli expelled H+ with low flux compared with that of glucose fermentation. The results indicate that at pH 5.5, when F1Fo activity is low [28], Hyd-1 or Hyd-2, but not the other Hyd enzymes, may participate in H+ extrusion or have H+-translocating ability.

EXPERIMENTAL

Bacterial strains growth and membrane vesicles preparation

The E. coli wild-type and mutant strains used are listed in Table 1. All strains were supplied by Professor T.K. Wood (Texas A&M University, College Station, TX, U.S.A.). Bacteria were grown under anaerobic conditions at 37°C in a peptone medium (20 g/l peptone, 15 g/l K2HPO4 and 10 g/l NaCl) with 10 g/l glycerol (pH 7.5 and 6.0). The pH was measured with a pH meter with a selective pH electrode (HJ1131B; Hanna Instruments) and adjusted by means of 0.1 M NaOH or 0.1 M HCl. Membrane vesicles were prepared as described by Kirakosyan et al. [30].

Table 1
Characteristics of the E. coli strains used

Abbreviation: KmR, kanamycin resistance.

StrainGenotypeAbsent or defective appropriate proteinReference
BW25113 lacl rrnBT14 ΔlacZW116 hsdR514 ΔaraBADAH33 Δrha BADLD78 Wild-type [13
JW0955 BW25113 ΔhyaB Δkan Large subunit of Hyd-1 [13
JW2701 BW 25113 ΔfhlA KmR FHL activator [29
JW2962 BW 25113 ΔhybC Large subunit of Hyd-2 [13
MW1000 BW25113 ΔhyaB hybC Large subunits of Hyd-1 and Hyd-2 [13
StrainGenotypeAbsent or defective appropriate proteinReference
BW25113 lacl rrnBT14 ΔlacZW116 hsdR514 ΔaraBADAH33 Δrha BADLD78 Wild-type [13
JW0955 BW25113 ΔhyaB Δkan Large subunit of Hyd-1 [13
JW2701 BW 25113 ΔfhlA KmR FHL activator [29
JW2962 BW 25113 ΔhybC Large subunit of Hyd-2 [13
MW1000 BW25113 ΔhyaB hybC Large subunits of Hyd-1 and Hyd-2 [13

Determination of ion fluxes

H+ and K+ fluxes by whole cells were determined by monitoring the changes in H+ and K+ activities in the medium with the use of selective pH (Hanna Instruments) and K+ electrodes (Cole Parmer Instruments) [17,21,23]. The electrode readings were calibrated by titration of the medium with small quantities of 0.01 M HCl and 0.01 M KCl. Ion fluxes were expressed in mmol/min per number of cells in 1 unit of volume.

ATPase assay

ATPase activity in membrane vesicles was calculated by the quantity of Pi produced in the reaction with 3 mM ATP [31]. The assay medium was 50 mM Tris/HCl containing 2.5 mM MgSO4 and 100 mM KCl (if not mentioned otherwise). Pi was determined colorimetrically with a Spectro UV–VIS Auto PC scanning spectrophotometer (LaboMed). ATPase activity was expressed in μmol of Pi/min per mg of protein.

Other procedures and data processing

The attenuance at 600 nm of the bacterial culture was measured with a spectrophotometer. The number of cells in 1 unit of volume was counted as the number of colonies after re-plating the diluted bacterial suspension on solid nutrient medium. Protein levels were measured by the method of Lowry et al. [32] using BSA as a standard. For DCCD inhibition studies, whole cells or vesicles were incubated with 0.2 mM DCCD for 10 min. All assays were done at 37°C. Data were averaged from duplicate or triplicate independent measurements, for which the S.E.M. does not exceed 3% (if not indicated otherwise). For the difference between the values, a Student's t test for validity criteria (P) was calculated; it was <0.01 (if not mentioned otherwise) [33].

RESULTS AND DISCUSSION

H+ efflux by whole cells and ATPase activity of membrane vesicles of glycerol-fermented E. coli at acidic and slightly alkaline pH

E. coli grown under anaerobic conditions during fermentation of glucose or other sugars (lactose or maltose) at alkaline pH has been demonstrated to acidify the medium and accumulate K+ with the fixed (2H+/K+) stoichiometry of the initial DCCD-sensitive fluxes via F1Fo and TrkA respectively, as well as to produce H2 by FHL [18,23,34]. It was shown that FHL is required for H+/K+ exchange: most probably it serves to supply reducing equivalents for energy transfer to facilitate K+ uptake via TrkA [18,23,26]. Although FHL functioning and H2 production require F1Fo in glucose-fermented cells, little information is available about H2 production and its energetics, particularly upon glycerol fermentation.

The kinetics of H+ and K+ fluxes by E. coli BW25113 whole cells grown under glycerol or glucose fermentation at pH 7.5 is illustrated in Figure 1. H+ and K+ fluxes were quite different for bacteria in the presence of glycerol or glucose: both the fluxes were lower in glycerol-fermented cells than those for cells grown under glucose fermentation. Separate experiments were done with DCCD (0.2 mM), F1Fo and other H+ translocation mechanism inhibitors [19,20]. There was ~1.3-fold inhibition of H+ efflux by DCCD on glycerol fermentation (Figure 2), suggesting the participation of F1Fo in H+ secretion. H+ release by E. coli BW25113 was also investigated at pH 5.5 under glycerol fermentation (Figure 2): the H+ efflux was ~3 times lower compared with that at pH 7.5.

Kinetics of H+/K+ exchange by E. coli wild-type BW25113 during fermentation of glycerol and glucose at pH 7.5

Figure 1
Kinetics of H+/K+ exchange by E. coli wild-type BW25113 during fermentation of glycerol and glucose at pH 7.5

Bacteria were grown at the initial pH 7.5 under glycerol (Gly, black symbols) or glucose (Gl, white symbols) fermentation, washed in distilled water and transferred to 150 mM Tris/phosphate buffer (pH 7.5) containing 0.4 mM MgSO4, 1 mM KCl, 1 mM NaCl and either 10 g/l glycerol or 20 mM glucose. For both cases, the bacterial count was 1010 cells/ml.

Figure 1
Kinetics of H+/K+ exchange by E. coli wild-type BW25113 during fermentation of glycerol and glucose at pH 7.5

Bacteria were grown at the initial pH 7.5 under glycerol (Gly, black symbols) or glucose (Gl, white symbols) fermentation, washed in distilled water and transferred to 150 mM Tris/phosphate buffer (pH 7.5) containing 0.4 mM MgSO4, 1 mM KCl, 1 mM NaCl and either 10 g/l glycerol or 20 mM glucose. For both cases, the bacterial count was 1010 cells/ml.

H+ efflux by whole cells of E. coli wild-type and fhlA, hyaB, hybC and hyaB hybC mutant strains during glycerol fermentation at pH 7.5 and 5.5

Figure 2
H+ efflux by whole cells of E. coli wild-type and fhlA, hyaB, hybC and hyaB hybC mutant strains during glycerol fermentation at pH 7.5 and 5.5

Bacteria grown at pH 7.5 and 5.5 were assayed at pH 7.5 and 5.5 respectively. A parallel experiment was performed in the presence of 0.2 mM DCCD. For mutant strains, see Table 1; for the others, see the Experimental section and the legend to Figure 1.

Figure 2
H+ efflux by whole cells of E. coli wild-type and fhlA, hyaB, hybC and hyaB hybC mutant strains during glycerol fermentation at pH 7.5 and 5.5

Bacteria grown at pH 7.5 and 5.5 were assayed at pH 7.5 and 5.5 respectively. A parallel experiment was performed in the presence of 0.2 mM DCCD. For mutant strains, see Table 1; for the others, see the Experimental section and the legend to Figure 1.

Here, ATPase activity was investigated with membrane vesicles of glycerol-fermented E. coli BW25113 at pH 7.5: this activity was strongly inhibited by 0.2 mM DCCD (Figure 3). The ATPase activity was ~2-fold lower at pH 7.5 compared with the ATPase activity of vesicles of cells grown under glucose fermentation (results not shown). Interestingly, the ATPase activity value was in correlation with low H+ efflux by E. coli under glycerol fermentation. Moreover, there was no detectable stimulation of ATPase activity by 100 mM K+ (results not shown). The results may be explained assuming that this activity was not dependent on TrkA under glycerol fermentation at pH 7.5.

ATPase activity of membrane vesicles of E. coli wild-type and fhlA, hyaB, hybC and hyaB hybC mutant strains at pH 7.5 and 5.5

Figure 3
ATPase activity of membrane vesicles of E. coli wild-type and fhlA, hyaB, hybC and hyaB hybC mutant strains at pH 7.5 and 5.5

The amount of membrane protein in the assay sample was 0.06–0.1 mg; for the others, see the Experimental section and the legend to Figure 1.

Figure 3
ATPase activity of membrane vesicles of E. coli wild-type and fhlA, hyaB, hybC and hyaB hybC mutant strains at pH 7.5 and 5.5

The amount of membrane protein in the assay sample was 0.06–0.1 mg; for the others, see the Experimental section and the legend to Figure 1.

Thus the H+ efflux and the K+ influx by E. coli whole cells as well as the ATPase activity of membrane vesicles were first demonstrated to be lower during glycerol fermentation at pH 7.5 than that for cells fermenting glucose; F1Fo participated in H+ efflux and was probably operated independently of TrkA.

H+ efflux and ATPase activity of glycerol-fermented E. coli Hyd mutants strains at slightly alkaline and acidic pH

To reveal the role of different Hyd enzymes in H+ efflux and F1Fo activity, E. coli hyaB and hybC mutant strains with defective Hyd-1 and Hyd-2 respectively, hyaB hybC double mutant with defective Hyd-1 and Hyd-2, and a mutant with deletion of the fhlA gene, coding for the transcriptional activator for Hyd-3 and Hyd-4 (see Table 1), were investigated at pH 7.5 and 5.5. The H+ efflux in the hyaB, hybC and hyaB hybC mutants compared with that in wild-type was noticeably lower (Figure 2). In contrast with the other mutants, significantly high H+ efflux was observed in the fhlA mutant. DCCD inhibited H+ efflux by ~2-fold with hyaB, ~1.2-fold (P<0.05) with hybC, ~2.2-fold with hyaB hybC and ~1.8-fold with fhlA mutants (Figure 2). Regarding the K+ accumulation by these mutants, the latter was less, almost absent (results not shown).

H+ efflux was also examined at pH 5.5 with all the mutants mentioned above (see Figure 2). As shown for the E. coli wild-type during glycerol fermentation, the H+ efflux was lower compared with that at pH 7.5. Here, the H+ efflux in hyaB and hybC mutants was similar to each other (see Figure 2). Again, H+ efflux was increased markedly in the fhlA mutant and absent in the hyaB hybC double mutant compared with that in wild-type (see Figure 2).

The results obtained may be explained as follows: at neutral and slightly alkaline pH under glycerol fermentation, Hyd-1 and Hyd-2 could have a relationship with F1Fo or participate in H+ translocation through the membrane. The stimulation of H+ efflux in the fhlA mutant might indicate that here, upon glycerol fermentation, Hyd-3 and Hyd-4 could operate in H+ uptake mode; this is in agreement with results suggesting that both Hyd-3 and Hyd-4 probably have H+-translocating ability [12,16,25]. The results are also consistent with the results suggesting that upon glucose fermentation, Hyd-3 might be operating in H2 uptake mode but not in producing mode [13,14], while, on the other hand, upon glycerol fermentation, Hyd-1 and Hyd-2 function in a reverse mode [14]. Moreover, the results may suggest that, at acidic pH during glycerol fermentation, both Hyd-1 and Hyd-2 more than F1Fo might be involved in H+ efflux.

Membrane vesicle ATPase activity at pH 7.5, compared with that in wild type cells, was lower by ~2-fold with hyaB and ~20-fold with hybC mutants (Figure 3). It was stimulated markedly (~1.5 times) in the hyaB hybC double mutant and was suppressed significantly (~3 times) in the fhlA mutant (see Figure 3). The markedly decreased total and DCCD-inhibited ATPase activity at pH 7.5 in hyaB and hybC mutants leads to the suggestion that Hyd-2 more than Hyd-1 might have a close relationship with F1Fo. Interestingly, the stimulation of ATPase activity in the hyaB hybC double mutant and the suppression in hyaB and hybC mutants at pH 7.5 might be explained by interaction between different Hyd enzymes (Hyd-1 and Hyd-2) as well as their interactions with FoF1. A similar situation with the deletion effects of different genes coding for the K+ transport systems to change K+ transport activity in E. coli was discussed by Dosch et al. [35] and in previous papers from our laboratory [21,31,34]. DCCD at the same concentration as that used with the H+ efflux study inhibited ATPase activity by ~2-fold with hyaB, ~10-fold (P<0.001) with hybC, ~3-fold with hyaB hybC and ~2.3-fold with fhlA mutants (Figure 3).

However, at pH 5.5, the ATPase activities of the hyaB and hybC mutants had similar values and were higher compared with that in wild-type; ATPase activity was suppressed by ~1.5-fold in hyaB hybC and ~6-fold in fhlA mutants (Figure 3). The low ATPase activity of the fhlA mutant is in agreement with the suggestion that FhlA coded for by this gene has an ATPase activity [25,36,37]. Moreover, the absence of any DCCD effect on the ion fluxes by E. coli whole cells and ATPase activities of membrane vesicles at pH 5.5 observed is in accord with a previous study indicating that this reagent did not affect cells at low pH [28].

Concluding remarks

During glycerol fermentation by E. coli at slightly alkaline pH, the H+ efflux and K+ accumulation rates of whole cells as well as ATPase activity of membrane vesicles are lower than those for cells fermenting glucose: F1Fo participates in H+ efflux and probably operates independently of TrkA. At pH 7.5, Hyd-1 and Hyd-2 could have a close relationship to F1Fo or participate in H+ translocation, whereas Hyd-3 and Hyd-4 might operate in H+ uptake mode. At pH 5.5, Hyd-1 and Hyd-2 more than F1Fo might be involved in H+ efflux.

The results presented above point to the relationship of different Hyd enzymes to F1Fo during glycerol fermentation playing an important role in generation of ΔμH+, detoxification of the formic acid formed during fermentation, and neutralization of the cytoplasm.

Abbreviations

     
  • DCCD

    dicyclohexylcarbodi-imide

  •  
  • Fdh-H

    formate dehydrogenase H

  •  
  • FHL

    formate hydrogen lyase in Escherichia coli

  •  
  • Hyd

    hydrogenase

  •  
  • Hyd-1

    Hyd-2, Hyd-3 or Hyd-4, the first, second, third or fourth Hyd in E. coli

  •  
  • ΔμH+

    protonmotive force

AUTHOR CONTRIBUTION

Syuzanna Blbulyan carried out the ATPase activity assays (bacterial growth, isolation of membrane vesicles, determination of protein in membrane vesicles and ATPase activity). Arev Avagyan determined the ions fluxes through the bacterial membrane (bacterial growth, isolation of membrane vesicles, determination of cell numbers and ion fluxes). Anna Poladyan analysed and processed the experimental data, and wrote the manuscript. Armen Trchounian supervised, led discussions, and re-wrote and revised the manuscript.

We thank Professor T.K. Wood for supplying E. coli strains and for valuable advice.

FUNDING

This study was supported by the Ministry of Education and Science of the Republic of Armenia [grant number 1018-2008].

References

References
1
Bock
 
A.
Sawers
 
G.
 
Neidhardt
 
F. G.
Curtis
 
R.
Ingroham
 
J. L.
Lin
 
E. C. C.
Low
 
K. B.
Magasanik
 
B.
Reznikoff
 
W. S.
Riey
 
M.
Schaechter
 
M.
Umbarger
 
H. E.
 
Fermentation
Escherichia coli and Salmonella: Cellular and Molecular Biology
1996
Washington, DC
ASM Press
(pg. 
262
-
282
)
2
Dharmadi
 
Y.
Murarka
 
A.
Gonzalez
 
R.
 
Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering
Biotechnol. Bioeng.
2006
, vol. 
94
 (pg. 
821
-
828
)
3
Booth
 
I. R.
 
Bock
 
A.
 
Glycerol and methylglyoxal metabolism
EcoSal – Escherichia coli and Salmonella: Cellular and Molecular Biology
2006
Washington, DC
ASM Press
 
chapter 3.4.3
4
Poladyan
 
A.
Trchounian
 
A.
 
Trchounian
 
A.
 
Production of molecular hydrogen by mixed-acid fermentation in bacteria and its energetics
Bacterial Membranes
2009
Trivandrum, Kerala, India
Research Signpost
(pg. 
197
-
231
)
5
Abhishek
 
M.
Dharmadi
 
Y.
Yazmandi
 
S. S.
Gonzalez
 
R.
 
Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals
Appl. Environm. Microbiol.
2008
, vol. 
74
 (pg. 
1124
-
1135
)
6
Sawers
 
R. G.
 
Formate and its role in hydrogen production in Escherichia coli
Biochem. Soc. Trans.
2005
, vol. 
33
 (pg. 
42
-
46
)
7
Hartel
 
U.
Buckel
 
W.
 
Sodium ion-dependent hydrogen production in Acidaminococcus fermentans
Arch. Microbiol.
1996
, vol. 
166
 (pg. 
350
-
356
)
8
Sasahara
 
K. C.
Heinzinger
 
N. K.
Barrett
 
E. L.
 
Hydrogen sulfide production and fermentative gas production by Salmonella typhimurium require F1Fo ATP synthase activity
J. Bacteriol.
1997
, vol. 
179
 (pg. 
6736
-
6740
)
9
Steuber
 
J.
Krebs
 
W.
Bott
 
M.
Dimroth
 
P.
 
A membrane-bound NAD(P)+- reducing hydrogenase provides reduced pyridine nucleotides during citrate fermentation by Klebsiella pneumonia
J. Bacteriol.
1999
, vol. 
181
 (pg. 
241
-
245
)
10
Ballantine
 
S. P.
Boxer
 
D. H.
 
Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli
Eur. J. Biochem.
1986
, vol. 
156
 (pg. 
277
-
284
)
11
Sauter
 
M.
Bohm
 
R.
Bock
 
A.
 
Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli
Mol. Microbiol.
1992
, vol. 
6
 (pg. 
1523
-
1532
)
12
Andrews
 
S. C.
Berks
 
B. C.
McClay
 
J.
Ambler
 
A.
Quail
 
M. A.
Golby
 
P.
Guest
 
J. R.
 
A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-translocating formate-hydrogenlyase system
Microbiology
1997
, vol. 
143
 (pg. 
3633
-
3647
)
13
Maeda
 
T.
Sanchez-Torres
 
V.
Wood
 
T. K.
 
Enhanced hydrogen production from glucose by metabolically engineered Escherichia coli
Appl. Microbiol. Biotechnol.
2007
, vol. 
76
 (pg. 
1036
-
1042
)
14
Trchounian
 
K.
Trchounian
 
A.
 
Hydrogenase 2 is most and hydrogenase 1 is less responsible for H2 production by Escherichia coli under glycerol fermentation at neutral and slightly alkaline pH
Int. J. Hydrogen Energy
2009
, vol. 
34
 (pg. 
8839
-
8845
)
15
Bagramyan
 
K.
Mnatsakanyan
 
N.
Poladian
 
A.
Vassilian
 
A.
Trchounian
 
A.
 
The roles of hydrogenases 3 and 4, and the F1Fo-ATPase, in H2 production by Escherichia coli at alkaline and acidic pH
FEBS Lett.
2002
, vol. 
516
 (pg. 
172
-
178
)
16
Hakobyan
 
M.
Sargsyan
 
H.
Bagramyan
 
K.
 
Proton translocation coupled to formate oxidation in anaerobically grown fermenting Escherichia coli
Biophys. Chem.
2005
, vol. 
115
 (pg. 
55
-
61
)
17
Bagramyan
 
K. A.
Martirosov
 
S. M.
 
Formation of an ion transport supercomplex in Escherichia coli. An experimental model of direct transduction of energy
FEBS Lett.
1989
, vol. 
249
 (pg. 
149
-
152
)
18
Trchounian
 
A.
Bagramyan
 
K.
Poladyan
 
A.
 
Formate hydrogenlyase is needed for proton-potassium exchange through the F1Fo-ATPase and the TrkA system in anaerobically grown and glycolysing Escherichia coli
Curr. Microbiol.
1997
, vol. 
35
 (pg. 
201
-
206
)
19
Azzi
 
A.
Casey
 
R. P.
Nalecz
 
M. J.
 
The effect of N,N′-dicyclohexylcarbodiimide on enzymes of bioenergetic relevance
Biochim. Biophys. Acta
1984
, vol. 
768
 (pg. 
209
-
226
)
20
Hassinen
 
I. E.
Vuokila
 
P. T.
 
Reaction of dicyclohexylcarbodiimide with mitochondrial proteins
Biochim. Biophys. Acta
1993
, vol. 
1144
 (pg. 
107
-
123
)
21
Martirosov
 
S. M.
Trchounian
 
A. A.
 
An electrochemical study of energy-dependent potassium accumulation in E. coli. 10. Operation of H+–K+-exchanging mechanisms in unc mutants
Bioelectrochem. Bioenerg.
1983
, vol. 
11
 (pg. 
29
-
36
)
22
Martirosov
 
S. M.
Trchounian
 
A. A.
 
An electrochemical study of energy-dependent potassium accumulation in E. coli. 11. The Trk system in anaerobically and aerobically grown cells
Bioelectrochem. Bioenerg.
1986
, vol. 
15
 (pg. 
417
-
426
)
23
Trchounian
 
A.
Ohanjanyan
 
Y.
Bagramyan
 
K.
Zakharyan
 
E.
Vardanian
 
V.
Vassilian
 
A.
Davtian
 
M.
 
Relationship of the Escherichia coli TrkA system of potassium ion uptake with the F1Fo-ATPase under growth conditions without anaerobic or aerobic respiration
Biosci. Rep.
1998
, vol. 
18
 (pg. 
143
-
154
)
24
Nakamoto
 
R. K.
Ketchum
 
C. J.
Kuo
 
P. H.
Peskova
 
Y. B.
Al-Shawi
 
M.
 
Molecular mechanism of rotational catalysis in the F1Fo-ATP synthase
Biochim. Biophys. Acta
2000
, vol. 
1458
 (pg. 
289
-
299
)
25
Bagramyan
 
K.
Trchounian
 
A.
 
Structure and functioning of formate hydrogen lyase
Biochemistry (Moscow)
2003
, vol. 
68
 (pg. 
1159
-
1170
)
26
Trchounian
 
A.
 
Escherichia coli proton-translocating F1Fo-ATP synthase and its association with solute secondary transporters and/or enzymes of anaerobic oxidation-reduction under fermentation
Biochem. Biophys. Res. Commun.
2004
, vol. 
315
 (pg. 
1051
-
1057
)
27
Blokesch
 
M.
Magalon
 
A.
Bock
 
A.
 
Interplay between the specific chaperone-like proteins HybG and HypC in maturation of hydrogenases 1, 2 and 3 from Escherichia coli
J. Bacteriol.
2001
, vol. 
189
 (pg. 
2817
-
2822
)
28
Trchounian
 
A.
Kobayashi
 
H.
 
Kup is the major K+ uptake system in Escherichia coli upon hyper-osmotic stress at low pH
FEBS Lett.
1999
, vol. 
447
 (pg. 
144
-
148
)
29
Baba
 
T.
Ara
 
T.
Hasegawa
 
M.
Takai
 
Y.
Baba
 
M.
Datsenko
 
K. A.
Tomita
 
M.
Wanner
 
B. L.
Mori
 
H.
 
Construction of Escherichia coli K-12 in-frame, single gene knockout mutants: the Keio Collection
Mol. Syst. Biol.
2006
 
2.2006.0008
30
Kirakosyan
 
G.
Trchounian
 
K.
Vardanyan
 
Z.
Trchounian
 
A.
 
Copper(II) ions affect Escherichia coli membrane vesicles' SH-groups and a disulfide–dithiol interchange between membrane proteins
Cell Biochem. Biophys.
2008
, vol. 
51
 (pg. 
45
-
50
)
31
Trchounian
 
A.
Vassilian
 
A.
 
Relationship between the F1Fo-ATPase and K+ transport system within the membrane of anaerobically grown Escherichia coli. N,N′-dicyclohexylcarbod iimide-sensitive ATPase activity in trk mutants
J. Bioenerg. Biomembr
1994
, vol. 
26
 (pg. 
563
-
571
)
32
Lowry
 
O. H.
Rosebrogh
 
N. J.
Farr
 
A. L.
Randall
 
R. J.
 
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
1953
, vol. 
193
 (pg. 
265
-
275
)
33
Lakin
 
G. F.
 
Biometry
1992
Moscow
Vishaya Shcola
34
Trchounian
 
A.
 
Trchounian
 
A.
 
Potassium transport by bacteria: electrochemical approach, energetic and mechanisms
Bacterial Membranes
2009
Trivandrum, Kerala, India
Research Signpost
(pg. 
65
-
111
)
35
Dosch
 
D. C.
Helmer
 
G. L.
Sutton
 
S. H.
Salvacion
 
F. F.
Epstein
 
W.
 
Genetic analysis of potassium transport loci in Escherichia coli: evidence for three constitutive systems mediating uptake of potassium
J. Bacteriol.
1991
, vol. 
173
 (pg. 
687
-
696
)
36
Leonhartsberger
 
S.
Korsa
 
I.
Bock
 
A.
 
The molecular biology of formate metabolism in enterobacteria
J. Mol. Microbiol. Biotechnol.
2002
, vol. 
4
 (pg. 
269
-
276
)
37
Hopper
 
S.
Bock
 
A.
 
Effector-mediated stimulation of ATPase activity by the sigma 54-dependent transcriptional activator FHLA from Escherichia coli
J. Bacteriol.
1995
, vol. 
177
 (pg. 
1798
-
1803
)