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Keywords:

  • Salinibacter ruber;
  • Glutamate dehydrogenase;
  • Halophilic enzyme

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Salinibacter ruber, an extremely halophilic member of the domain Bacteria, has two different cytoplasmic glutamate dehydrogenase activities, marked as GDHI and GDHII. GDHI showed a strong dependence on high salt concentrations for stability, but not for activity, displaying maximal activity in the absence of salts. GDHII depended on high salt concentrations for both activity and stability. It catalyzed amination of 2-oxoglutarate with optimal activity in 3 M KCl at pH 8. No activating effect was found when NaCl was replaced by KCl. Only GDHII displayed activity in the deamination reaction of glutamate with an optimal pH of 9.5. Both enzymes were activated by certain amino acids (l-leucine, l-histidine, l-phenylalanine) and by nucleotides such as ADP or ATP. A low-molecular-mass cytoplasmic fraction was found to be a highly effective activator of GDHII in the presence of high NaCl concentrations.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Salinibacter ruber is a red, rod-shaped, aerobic bacterium, phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria. It was first isolated from saltern crystallizer ponds in Spain [1]. It is an obligate halophile that requires at least 150 g l−1 salts for growth, and grows optimally between 200 and 300 g l−1 salts.

No significant concentrations of organic osmotic solutes, such as are accumulated by other aerobic halophilic representatives of the Bacteria [2,3], were found within S. ruber cells. The physiology of S. ruber resembles that of the Archaea of the family Halobacteriaceae: it accumulates molar concentrations of K+ intracellularly, intracellular Cl concentrations are high as well [4], and the bulk protein showed a great excess of acidic amino acids, a low content of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine [4], all indicators for the presence of a salt-adapted enzymatic machinery.

Many of the enzymes of S. ruber are truly halophilic, requiring salt for activity and/or stability. Examples are the NAD-dependent isocitrate dehydrogenase [5], the fatty acid synthetase complex [6], and the NADP-linked glucose-6-phosphate dehydrogenase [7]. Other enzymes such as the NADP-dependent isocitrate dehydrogenase [5] and glycerol kinase (Sher, Mana and Oren, submitted) may function as well in the presence of high salt and in its absence. Still others, such as the NAD-dependent malate dehydrogenase [5] and hexokinase [7], function best in a low salt environment and are inhibited by high salt concentrations. The behavior of the individual enzymes toward salt thus varies considerably.

l-Glutamate dehydrogenases (GDH) (EC 1.4.1.2–4) are important enzymes in any cell due to the pivotal position occupied by glutamate and 2-oxoglutarate in the central metabolism of nitrogen and carbon compounds. They catalyze the interconversion of 2-oxoglutarate and l-glutamate using NAD(H) or NADP(H) as cofactors. These enzymes are evolutionarily conserved in the three domains, Archaea, Bacteria, and Eucarya. GDHs can be differentiated on the basis of their molecular properties, their coenzyme specificity, and the regulation of their activity by purine nucleoside di- and triphosphates as well as other ligands [8,9].

Preliminary assays of NAD-dependent GDH, as assayed by the reductive amination of 2-oxoglutarate, showed an unusual behavior: in the absence of salt low activity was obtained, which further decreased with increasing KCl concentration, and no activity was found above 2.5 M KCl. However, a specific stimulation by high concentrations of NaCl was observed, with optimum activity at 3–3.5 M [5]. The present study was initiated to investigate the basis of this intriguing behavior. We here report the presence of two different GDH enzymes in S. ruber. They differ in their affinity for substrates, in their pH and salt dependence for activity and stability, and in their regulation by different effectors.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Bacterial strain and culture conditions

S. ruber strain M31 (DSM 13855T) was grown on a rotatory shaker in 2-l Erlenmeyer flasks containing 1 l of medium of the following composition (all concentrations in g l−1): NaCl, 195; MgSO4·7H2O, 25; MgCl2·6H2O, 16.3; CaCl2·2H2O, 1.25; KCl, 5.0; NaHCO3, 0.25; NaBr, 0.625; yeast extract, 1.0, pH 7.2. Late exponential phase cells were harvested by centrifugation (30 min, 9600×g) and suspended in 50 mM phosphate buffer (pH 6.6) containing 2.5 M (NH4)2SO4. Cells were broken by sonication, and debris was removed by centrifugation (60 min, 105 000×g, 4°C).

2.2Enzyme purification and characterization

Cell extract (80 ml, 336 mg protein) was applied to a Sepharose 4B column (2.5×30 cm), and eluted using an (NH4)2SO4 gradient from 2.5 M to 0.5 M in 20% (w/v) glycerol, and 10-ml fractions were collected and assayed for GDH activity. Fractions displaying salt-sensitive and salt-dependent GDH activity, assayed as described in Section 2.3, were pooled separately and marked as GDHI and GDHII. Each pool was concentrated using a DEAE-cellulose column (3×5 cm), eluted with 20 mM Tris–HCl buffer pH 8.0 containing 3 M NaCl, and was further purified by gel filtration on a Sepharose CL-6B column. The protein content of the fractions was determined by the Bradford method [10].

Samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the apparent molecular mass of the native GDH fractions was determined by fast performance liquid chromatography (FPLC, Pharmacia), by applying a sample of each GDH on a HiPrep Sephacryl S-300 column (Pharmacia), previously equilibrated with 50 mM Tris–HCl buffer pH 7.3 containing 2 M NaCl, and calibrated with different proteins ranging in size from 425 kDa (ferritin) to 12.5 kDa (cytochrome c) as molecular mass markers.

2.3GDH activity assays

GDH was assayed according to both the amination reaction (reductive amination of 2-oxoglutarate) and the deamination reaction (oxidative deamination of l-glutamate). The reaction mixture, in a final volume of 2 ml, for the amination reaction typically contained 40 μmol 2-oxoglutarate, 0.6 μmol NADH or NADPH, 0.2 mmol ammonium acetate, all of them dissolved in a 20 mM Tris–HCl buffer, pH 8.0 and containing NaCl and/or KCl as indicated. The reaction was started by the addition of 2-oxoglutarate. Reaction mixtures for the deamination reaction typically contained 100 μmol l-glutamate, 6.0 μmol NAD+ or NADP+, dissolved in 20 mM Tris–HCl, pH 8.0 and NaCl and/or KCl as indicated, in a final volume of 2 ml. The oxidation of NAD(P)H or the reduction of NAD(P) was followed at 40°C at 340 nm in a Ultrospec 2000 spectrophotometer (Pharmacia Biotech), provided with a temperature-controlled cuvette holder. For determination of the pH optima, assays were performed in 50 mM phosphate buffer (pH range 5–7), 50 mM Tris–HCl (pH range 7–9), and 50 mM CHES buffer for the pH range 9–10.5. When indicated, different effectors such as l-histidine, l-glutamine, l-leucine, ADP and ATP were added to the reaction mixtures. The maximum velocity (Vmax) and the Michaelis constant (Km) for different substrates were calculated from the reciprocal initial velocities plotted versus reciprocal substrate concentrations, using the algorithm of Marquardt–Levenberg with the SigmaPlot program (Jandel Scientific, v. 1.02).

2.4Enzyme stability tests

For stability assays, highly concentrated stocks of each enzyme were used. They were diluted to different salt concentrations as indicated in 20 mM Tris–HCl buffer, pH 8.0, at 20°C. Samples were taken at different time intervals, and the GDH activity was determined under optimal conditions for each enzyme. The logarithm of the relative activity as compared to the initial rate obtained was plotted as a function of time to determine the pseudo-first-order constant for the time-dependent loss of activity and to calculate the half-life of the enzyme at each salt concentration.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Purification of two GDHs from S. ruber

The purification protocol followed for S. ruber GDHs was very similar to that used earlier for GDHs from halophilic Archaea [9,11–13], indicating a similar halophilic behavior and probably similar properties. Two fractions with GDH activity were separated during chromatography of a cell extract on Sepharose 4B (Fig. 1). The first peak, designated GDHII, was optimally active in Tris–HCl buffer, pH 8 in the presence of 4 M NaCl. The second, marked GDHI, showed optimal activity in Tris–HCl buffer pH 7.3 in the absence of salt. The two fractions were purified as indicated in Table 1 and tested individually for activity, stability, and other characteristics.

image

Figure 1. Purification of two GDHs from S. ruber on a Sepharose 4B column, using a decreasing (NH4)2SO4 gradient from 2.5 to 0.5 M as displayed by the straight line. The activity of fractions 260–280 (?) (GDHII) was measured in 20 mM Tris–HCl buffer containing 3 M NaCl, pH 8, including in the reaction mixture 100 μl of the pooled activating fractions (15–20). The activity of the second peak (fractions 285–290; GDHI) (▵) was determined in 20 mM Tris–HCl, pH 8 with no salt added.

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Table 1.  Description of the purification steps for GDH activities from S. ruber
  1. The samples from the second step were previously concentrated before application on a HiPrep (16/60) Sephacryl S-300 column.

Purification step Volume (ml)Activity (U ml−1)Protein (mg ml−1)Specific activity (U mg−1)Purification factor
Crude extract 800.484.20.11
Sepharose 4BGDHI400.120.0452.71
A.S. gradientGDHII500.090.024.51
Sephacryl S-300GDHI150.0390.00516.56
 GDHII180.330.01423.65

The apparent molecular masses of the native enzymes, as estimated by FPLC on a Sephacryl G200 column, were 226±44 kDa for GDHI and 460±120 kDa for GDHII. SDS–PAGE showed subunit sizes of 40±5 kDa for GDHI and 61±4 kDa for GDHII. A hexameric structure is thus suggested for GDHI, while GDHII may behave as an octamer or an aggregate of two tetramers. Bacterial and fungal NADP-dependent GDHs generally have a hexameric structure [8]. Such a structure was also reported for the archaeal NADP-dependent GDH from Haloferax mediterranei[13]. NAD-dependent GDHs have either four identical subunits, such as found in Neurospora crassa[14] or, more commonly, six [15].

3.2Substrate specificity, pH optimum and salt dependence

GDHI displayed activity in the reductive amination reaction only, and no significant activity of the deamination reaction could be shown. GDHII was active both in the reductive amination of 2-oxoglutarate and in the oxidative deamination of glutamate.

NADH was the sole electron donor for the reductive amination reaction by GDHI. GDHII showed dual cofactor specificity: NADPH can be used as electron donor, but at a lower maximal rate than with NADH, and the enzyme's affinity for NADPH is much lower than for NADH. Kinetic parameters of the two enzymes are presented in Table 2.

Table 2.  Kinetic parameters for GDH activities in S. ruber determined for the amination reaction in 50 mM Tris–HCl buffer, pH 8.0 containing 3 M KCl
Substrate Vmax (U ml−1)Km (mM)Vmax/Km
NADHGDHI0.38±0.040.19±0.042.0±0.2
 GDHII0.192±0.0110.064±0.0093.0±0.3
NADPHGDHINo activity
 GDHII0.29±0.070.42±0.081.40±0.09
2-OxoglutarateGDHI0.101±0.0115.6±0.70.018±0.03
 GDHII0.111±0.0137.1±0.70.016±0.002
NH4+GDHI0.36±0.09300±1000.0013±0.0003
 GDHII0.252±0.01368±80.0037±0.0003

GDHI was optimally active at pH 8 (in 50 mM Tris–HCl). The same optimal pH was found for the amination reaction of GDHII. The optimum pH of the deamination reaction varied with the assay conditions from 8.5 (50 mM Tris–HCl buffer containing 3 M KCl) to 9.5 (50 mM CHES buffer containing 3 M NaCl).

The two enzymes differed markedly in their response to salt. GDHI was optimally active in the absence of salt, and showed little activity at NaCl concentrations above 1 M. On the other hand, GDHII was found to be a salt-dependent enzyme, which was not active below 2 M NaCl, and displayed higher activity (2.8 U ml−1) at 4 M NaCl, the highest concentration tested (Fig. 2). NaCl was a much better activator of GDHII than KCl: activity at 3 M NaCl was 46-fold higher than at 3 M KCl. These findings explain the unusual salt dependence of NAD-dependent GDH activity (measured by the amination reaction) in crude cell extract of S. ruber, showing low activity in the absence of salt, a decrease with increasing salt concentration, followed by a sharp increase in activity at the highest concentrations of NaCl but not of KCl [5]. The first part of the curve can be attributed to activity of the salt-sensitive GDHI and the second part to the NaCl-activated GDHII.

image

Figure 2. Dependence of the activities of S. ruber GDHI (?) and GDHII (?) on NaCl concentration.

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Although only GDHII required salt for activity, both enzymes depended on salt for stability, and both were inactivated upon incubation in the absence of salt. Glycerol also stabilizes both enzymes to some extent, but less well than 0.75 M NaCl. In this respect both enzymes behave as truly halophilic enzymes, similar to most enzymes of the halophilic Archaea that accumulate KCl intracellularly [2]. Both enzymes lost their activity when dialyzed in salt-free buffer, and at least 0.5 M NaCl was needed to retain their long-term activity. GDHII displayed higher pseudo-first-order inactivation constants than GDHI at all salt concentrations tested, and consequently has a longer half-life at salt concentrations below 1 M (Table 3).

Table 3.  Pseudo-first-order kinetic constants and the half-life for the inactivation of S. ruber GDHI and GDHII
EnzymeSalt concentrationk (min−1)t1/2 (h)
GDHIno salts5.40×10−421
 0.25 M NaCl8.78×10−5132
 0.50 M NaCl8.87×10−61303
 0.75 M NaCl7.03×10−61643
 20% (v/v) glycerol1.25×10−6922
GDHIIno salts5.12×10−32.3
 0.25 M NaCl1.62×10−37.1
 0.50 M NaCl3.23×10−436
 0.75 M NaCl5.28×10−62190
 20% (v/v) glycerol3.1×10−5373

3.3Effect of different activators

GDHII was strongly activated by a cytoplasmic fraction that eluted early from the Sepharose 4B column ((NH4)2SO4 concentration near 2.5 M). The activator was pooled from fractions 15–20 of the same Sepharose 4B chromatography from which GDHI and GDHII were isolated. This activating pool was not excluded when it was loaded on a Sephadex G10 column, indicating a low molecular mass for the activator(s). On the other hand, when it was loaded on a DEAE-cellulose column part of the activating compounds passed through the column and another part was retained, being eluted with Tris–HCl buffer pH 7.3 containing 4 M NaCl. The fraction that was not retained activated both GDH enzymes, enabling 1.5-fold stimulation for GDHI and 16.8- and 14-fold stimulation of GDHII in the reductive amination reaction and oxidative deamination, respectively.

The stimulating effect of low-molecular-mass compounds may be due to certain amino acids or nucleoside phosphates. As shown in Table 4, the activity of both GDHI and GDHII was significantly enhanced by the amino acids l-leucine and l-histidine, as well as by ADP. l-glutamine (10 mM) gave a stimulation comparable to that obtained with l-histidine. ATP activated both GDHs less well than did ADP. Many of those compounds stimulating S. ruber GDH also activate other GDHs such as the enzyme from bovine liver [16]. The NAD-dependent GDH from the halophilic archaeon Halobacterium salinarum was also activated by amino acids, but ADP or ATP had no effect [17,18]. The S. ruber GDHI was strongly activated by l-leucine in the presence of KCl, while ADP had little effect. On the other hand, ADP displayed very high activating effect on GDHII in 3 M KCl. GDHII was much more active at high concentrations of NaCl (3 M) than at 3 M KCl, but in the presence of l-leucine or ADP the activity with KCl was similar to that with NaCl.

Table 4.  Effect of l-leucine (5 mM), l-histidine (10 mM) and ADP (2.5 mM) on the reductive amination activity of S. ruber GDHI and GDHII
 GDHI (U ml−1)GDHII (U ml−1)
 3 M NaCl3 M KClNo salts3 M NaCl3 M KClNo salts
Control0.033±0.0090.07±0.020.29±0.051.2±0.20.026±0.0090.018±0.004
l-Leu0.29±0.050.45±0.020.89±0.022.8±0.42.2±0.40.99±0.08
l-His0.14±0.020.10±0.021.4±0.32.0±0.20.032±0.0090.025±0.004
ADP0.20±0.020.12±0.030.18±0.023.2±0.42.0±0.52.48±0.05
ATP0.06±0.020.12±0.020.31±0.051.7±0.30.025±0.0050.024±0.005

3.4Final comments

It may be assumed that the different modes of regulation of activity of GDHI and GDHII by salt and organic effectors may be related to the different roles the two enzymes may play in the cell. Both enzymes appear to be NAD-linked, although GDHII showed a low affinity for NADP as well. GDHI probably acts as an aminating enzyme, while GDHII may be active in the deamination of glutamate, but it also showed high activity in the aminating reaction.

The occurrence of at least two different GDHs with different specificity and different roles was also reported for the halophilic archaeon Hbt. salinarum[11]. Three genes encoding GDHs (gdhA1, gdhA2 and gdhB) have been assigned in the genome of Halobacterium NRC1 [19] and in the genome of Halobacterium R1. Hbt. salinarum possesses both an NAD-dependent and an NADP-dependent enzyme. The NAD-dependent enzyme acts in both directions, but is thought to be mainly catabolically active in the oxidative deamination of glutamate, and is optimally active at 3.2 M NaCl or 0.8 M KCl [17]. The deamination reaction is inhibited above 2 M KCl, while 4 M NaCl allowed excellent activity. The amination reaction was inhibited by KCl concentrations above 0.8 M, while activity increased with NaCl concentration up to 4 M [9]. The NAD-dependent enzyme studied by Britton et al. [20], however, is still active in 4 M KCl. The NADP-specific enzyme, assayed by the reductive amination of 2-oxoglutarate, required at least 0.5 M KCl or NaCl, was optimally active in 1.6 M KCl, and still showed excellent activity at 4 M NaCl [11]. NADP-dependent GDH was also detected in Hfx. mediterranei. The hexameric enzyme functions optimally at 1–2 M KCl or NaCl, and is still almost fully active in 3.5 M KCl. In the absence of salt activity was about one third of that under optimum conditions [13].

The results presented in this work show that, in spite of minor differences, the GDHs of S. ruber show a halophilic behavior markedly similar to that of the GDHs of halophilic Archaea of the family Halobacteriaceae, organisms that, although phylogenetically unrelated, share with Salinibacter the mode of osmotic adaptation by accumulating high intracellular concentrations of inorganic ions (K+, Cl) rather than using organic solutes to provide osmotic balance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

This work was supported by funds from MCYT BIO2002-03197.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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