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Keywords:

  • Dimethylsulfoxide reductase;
  • Molybdate-dependent gene expression;
  • mop genes;
  • Rhodobacter

Abstract

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

Expression of the dimethylsulfoxide respiratory (dor) operon of Rhodobacter is regulated by oxygen, light intensity and availability of substrate. Since dimethylsulfoxide reductase contains a pterin molybdenum cofactor, the role of molybdate in the regulation of dor operon expression was investigated. In this report we show that the molybdate-responsive transcriptional regulator, MopB, and molybdate are essential for maximal dimethylsulfoxide reductase activity and expression of a dorA::lacZ transcriptional fusion in Rhodobacter capsulatus. In contrast, mop genes are not required for the expression of the periplasmic nitrate reductase or xanthine dehydrogenase in R. capsulatus under conditions of molybdenum sufficiency. This is the first report demonstrating a clear functional difference between the ModE homologues MopB and MopA in this bacterium. The results suggest that MopA is primarily involved in the regulation of nitrogen fixation gene expression in response to molybdate while MopB has a role in nitrogen fixation and dimethylsulfoxide respiration.


1Introduction

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

The pterin molybdenum cofactor (Moco) is found in a wide variety of oxomolybdenum enzymes from all three domains of life [1]. Bacterial oxomolybdenum enzymes have a major role in anaerobic respiration; formate dehydrogenase, nitrate reductases and S-oxide/N-oxide reductases all contain the bis(molybdopterin guanine dinucleotide)Mo (bis(MGD)Mo) form of Moco [1,2]. The assembly of all oxomolybdenum enzymes takes place in the bacterial cytoplasm since even those enzymes destined for the periplasm are secreted as holoenzymes via the tat (mtt) export pathway [3,4]. As a consequence molybdate uptake into the cell is a key element of the biogenesis of oxomolybdenum enzymes. The regulation of molybdate uptake into the cell has been described in Escherichia coli, Azotobacter vinelandii and Rhodobacter capsulatus (reviewed in [5]). All three bacteria possess a high affinity molybdate uptake system encoded by the modABCD genes. The expression of modABCD is repressed by a molybdate-responsive regulatory protein known as ModE in E. coli[6,7] and A. vinelandii[8]. In E. coli, modE is located upstream of the modABCD operon and is divergently transcribed [7] while in A. vinelandii modE is the first gene in the mod operon [8]. In R. capsulatus there are two homologues of ModE known as MopA and MopB [9]. mopA is the first gene in the mod operon while mopB is adjacent to mopA and is divergently transcribed (Fig. 1). In addition to a role in the regulation of the expression of the high affinity molybdate transporter, Mop/ModE proteins have been shown to be involved in the regulation of nitrogenase expression in R. capsulatus[9] and A. vinelandii[10]. In R. capsulatus it has been shown that MopA and MopB mediate the molybdate-dependent repression of anfA, a gene which encodes the activator of the expression of genes involved in the synthesis of the alternative nitrogenase (Fe-nitrogenase) [9]. Analysis of transcription initiation sites in the anfA promoter region identified a DNA sequence with dyad symmetry and similar sequences were identified in front of mopA, (R. capsulatus), modA (E. coli) and modE, modG, anfA and vnfA (A. vinelandii) [11]. DNase 1 protection experiments have shown that purified ModE binds to palindromes that are very similar to this consensus sequence in the modABCD promoter of E. coli[12,13].

image

Figure 1. Organisation of the mod-mop Gene Cluster in R. capsulatus[8]. Arrows show the direction of transcription initiated from the Gm interposon.

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In addition to negative regulation of gene expression it has been established that, in E. coli, ModE acts as a transcriptional activator of the hyc and nar operons [14]. Despite its obvious importance as a component of Moco the importance of molybdate in the transcriptional control of the expression of genes encoding oxomolybdenum enzymes has not been investigated in great detail. R. capsulatus and Rhodobacter sphaeroides possess a dimethylsulfoxide respiratory system (encoded by the dor operon) which is more closely related to the trimethylamine-N-oxide (TMAO) respiratory system of E. coli (encoded by the tor operon) than the DMSO respiratory system of E. coli (encoded by the dms operon) [15,16]. The dor operon is composed of at least three structural genes, dorC, dorD and dorA (which encodes the DMSO reductase structural gene). In this paper we describe experiments which investigated the role of molybdate and mop genes on the expression of DMSO reductase in R. capsulatus.

2Materials and methods

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

2.1Bacterial strains and growth conditions

R. capsulatus KS36, (B10S, ΔnifHDK::Spc) and mop mutants derived from it were used routinely during this study [9]. R423AI (mopA::Gm) and R423AII (mopA::Gm) are distinguished by the orientation of the gentamicin (Gm) interposon in the BamH1 site of this gene (Fig. 1). In strain R423AI the Gm interposon induces a non-polar mutation while in strain R423AII the Gm interposon exerts a polar effect on the expression of modABCD. R423BI (mopB::Gm) and R423BII (mopB::Gm) are similar insertional mutations in the mopB gene with the Gm interposon inserted in both orientations at the BamH1 site (Fig. 1). Strains R423CI (ΔmopAB::Gm) and R423CII (ΔmopAB::Gm) are distinguished by the orientation of the Gm interposon which substitutes for a 0.9 kb BamH1 fragment spanning mopA, mopB and their promoter regions (Fig. 1).

R. capsulatus strains were grown on RCV as previously described [17]. Where required, molybdate free medium was prepared as described [18]. Cell free extracts were prepared by harvesting overnight cultures at 10 000×g for 10 min at 4°C and washing once in 50 mM Tris–HCl pH 8. The washed cells were resuspended in 5 ml ice cold 50 mM Tris–HCl pH 8.0 per 50 ml culture and disrupted by passing twice through a French Pressure Cell at approximately 1.l×105 kN/m2. Large cell fragments and unbroken cells were removed by centrifugation at 25 000×g for 15 min at 4°C while the membrane and the soluble fractions were separated by centrifugation at 145 000×g for 1 h at 4°C.

2.2Molecular genetic and biochemical techniques

Chromosomal dorA::lacZ fusions in R. capsulatus strains were constructed as described previously [15]. Plasmid pFR400 harbouring the R. sphaeroides nap genes was a generous gift from Dr Conrado Moreno-Vivian and was transferred from E. coli S17-1 to R. capsulatus as previously described [19]. DMSO reductase and β-galactosidase were assayed as described [20] and [21] respectively. Nitrate reductase was assayed as described in [22] while xanthine dehydrogenase activity was measured as in [23]. Protein determination was performed using the bicinchoninic acid reagent with bovine serum albumin as a standard [24].

3Results

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

3.1The effect of molybdate concentration and mop mutations on DMSO reductase activity in R. capsulatus

The ability to use DMSO as an electron acceptor is a ubiquitous property of strains of R. capsulatus and this enabled us to investigate the effect of mop mutations on the expression of DMSO reductase in the R. capsulatus KS36 and the mop mutants which had previously been used in studies of the Mo-dependent repression of anfA gene expression [9]. Cells were grown phototrophically in the presence of DMSO and cell free extracts were assayed for DMSO reductase activity. In strain KS36 the activity of DMSO reductase increased as a function of molybdate concentration and reached a maximum value at >1 μM molybdate. In contrast, the activity of DMSO reductase in the mopB mutants (R423BI and R423BII) was low at all molybdate concentrations tested (Fig. 2a) suggesting that MopB had an essential role in the synthesis of DMSO reductase. Fig. 2b shows that in strain R423AI the level activity of DMSO reductase increased as a function of molybdate concentration as in the isogenic mop+ strain KS36. This suggested that high levels of DMSO reductase could be produced in cells which possessed active MopB only (strain R423AI), although a role for the Mod high affinity molybdate uptake system could not be ruled out since this system would be expressed by read-through from the Gm promoter (Fig. 1). Although strain R423AII was not expected to express the high affinity molybdate transporter because of a polar effect of the Gm interposon in mopA (Fig. 1) it was expected that high levels of molybdate might lead to a high activity of DMSO reductase. However, we found that even at concentrations of molybdate up to 1 mM no increase in DMSO reductase activity in strain R423AII was observed (Fig. 2b). In order to test whether the ModABCD transporter, as well as MopB, was necessary for the expression of DMSO reductase, the activity of this enzyme was measured in a modB mutant (strain R432I) [9]. It was observed that the increase in DMSO reductase as a function of molybdate was similar in the modB mutant and modB+ strain (data not shown).

image

Figure 2. The effect of molybdate concentration on DMSO reductase activity. (a) ▪-KS36; •-R423BI (mopB::Gm); ▴-R423BII (mopB::Gm). (b) ▪-KS36; •-R423AI (mopA::Gm); ▴-R423AII (mopB::Gm).

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3.2The effect of molybdate concentration and mop mutations on dorA::lacZ expression in R. capsulatus

The observation that both molybdate and MopB were required for maximal expression of DMSO reductase strongly suggested that at least one of the mop genes acted as a regulator of the transcription of the dorCDA operon. Strains with a dorA::lacZ chromosomal fusion ([ΦdorA::lacZ]) were constructed and the β-galactosidase activity of these transcriptional fusions was measured as a function of molybdate concentration (Fig. 3). The [ΦdorA::lacZ] expression in cell free extracts of strain KS36 increased with molybdate concentration (Fig. 3a). The results indicated that dorCDA operon expression was about 10-fold greater in molybdate-sufficient cells compared to cells on Mo free media. In contrast, the [ΦdorA::lacZ] expression in the MopA, MopB double mutants (R423CI and R423CII) was independent of molybdate concentration (Fig. 3a) and exhibited about the same level activity of β-galactosidase as found in strain KS36 at zero molybdate concentration.

image

Figure 3. The effect of molybdate on the expression of a chromosomal dorAlac transcriptional fusion in various mop genetic backgrounds. ▪-KS36; •-R423CI (ΔmopAB::Gm); ▴-R423CII (ΔmopAB::Gm). (b) ▪-KS36; •-R423BI (mopB::Gm); ▴-R423BII (mopB::Gm). (c) ▪-KS36; •-R423AI (mopA::Gm); ▴-R423AII (mopA::Gm).

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Fig. 3b shows that [ΦdorA::lacZ] expression in MopB mutants was low at all molybdate concentrations tested irrespective of the orientation of the Gm interposon. In contrast, Fig. 3c shows in the MopA mutants [ΦdorA::lacZ] expression in strain R423AI was similar to that found in the parental strain KS36, and this resembles the pattern of DMSO reductase activity for the two strains (Fig. 2b). Similarly, the level of expression of the dorA::lac fusion in strain R423AII was extremely low and, like the MopA, MopB double mutants (Fig. 3a), showed no effect of molybdate on the level of expression.

3.3Effect of mop mutations on the activity of periplasmic nitrate reductase and xanthine oxidase

The above observations prompted us to investigate whether mop mutations affected the activity of other oxomolybdenum enzymes in R. capsulatus. Cells were grown in RCV medium that contains sufficient molybdate for formation of the Moco in oxomolybdenum enzymes. The cytoplasmic enzyme xanthine dehydrogenase was assayed and its activity did not appear to be greatly affected in any of the mop mutants (Table 1). The R. capsulatus strain that was used in the above experiments does not possess a nitrate reductase. In order to determine whether expression of the periplasmic nitrate reductase was affected by mop mutations, R. capsulatus strains harbouring the nap gene cluster from R. sphaeroides[19] were used. The level of periplasmic nitrate reductase activity was not affected significantly by mop mutations in molybdate-replete medium (Table 1). Taken together, these data suggested that mop genes regulate the expression of DMSO reductase but not the expression of two other oxomolybdenum enzymes in R. capsulatus.

Table 1.  DMSO reductase (DMSOR), xanthine dehydrogenase (XDH) and periplasmic nitrate reductase (Nap) activity in mop mutants
 Specific activity (mU (mg protein)−1)
 KS36AIAIIBIBIICICII
DMSOR35.530.02.86.21.4NDND
XDH5.54.36.96.55.13.95.9
Nap47.537.346.545.937.765.136.0
The results shown above are the average from triplicate assays. The error for the DMSOR and Nap activity measurements was ±6% while for XDH it was ±5%.

4Discussion

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

R. capsulatus is the only bacterium which has been described which contains two ModE transcriptional regulators. However, it should be noted that MopA and MopB have only 50% sequence identity, suggesting that they may have functional differences within the cell. The data presented in this paper indicate that MopB is essential for the molybdate-dependent expression of the dorCDA operon in R. capsulatus. Since high concentrations of molybdate could not suppress the Dor-minus phenotype of the MopB mutants or the MopA, MopB double mutants, the data show that the lack of DMSO reductase expression is not due to a failure to express the Mod high affinity molybdate uptake system. In the case of the expression of the alternative nitrogenase from R. capsulatus, both MopA and MopB can mediate the repression of anfA[9]. The degree of repression by each Mop protein differs depending on whether the Mod molybdate uptake system is active, but even in the absence of this transporter a high level of molybdate will cause repression of anf expression. In contrast, we found that only MopB could facilitate high levels of expression of DMSO reductase. MopA was not able to act alone. This suggests that MopA is primarily associated with the regulation of the expression of the mod operon and, since the expression of this operon is under the control of the Ntr system, it is a component of the nitrogen fixation regulon of R. capsulatus[11]. It would be expected that the expression of the mod operon would be rather low under the ammonium-sufficient conditions that were used in this experiment. However, it did not appear that the failure to express DMSO reductase in the mopA mutant R423AII was due to a failure to express the Mod high affinity molybdate uptake system. If this had been the case then it would have been expected that addition of high levels of molybdate to strain R423AII would have restored DMSO reductase activity. Thus, the reason for the low level of DMSO reductase expression in strain R423AII is not clear. We have investigated the molybdate-dependent induction of DMSO reductase in strain R423BI (MopA present, MopB absent) grown under conditions (serine as N-source) where a higher level of expression of mopA occurs [9]. Under these conditions the level of DMSO reductase activity remained low (Solomon and McEwan, unpublished observations), suggesting that MopB is essential for the molybdate-dependent induction of DMSO reductase.

Although the dorCDA operon of R. capsulatus and R. sphaeroides and the torCAD operon of E. coli show strong similarities in their structural genes and in their promoter regions [15,25] there have been no reports that ModE regulates tor operon expression. In contrast, ModE has been shown to bind to the hyc and nar promoter of E. coli and enhance the transcription of narX and also the hyc operon [14]. A ModE-consensus binding site for positive regulation has been identified which differs from the consensus-binding site for repression [14]. Sequence analysis of the dorCDA operon and the dorR gene which is transcribed from the complementary strand in R. capsulatus has identified an intergenic region which contains the putative promoters for these operons [15]. In R. sphaeroides it has been shown that the response regulator, DorR, binds to this intergenic region and activates transcription of the dorCDA operon [25]. However, there are no sequences within this intergenic region that correspond to the Mop/ModE consensus sequence associated with positive regulation by ModE. Nevertheless, the data in this paper show that the molybdate/MopB-dependent expression of DMSO reductase has an effect on transcription of the dorCDA operon. This leads to the view that the positive regulation of dorCDA expression by MopB is indirect. It is possible that additional MopB-dependent regulatory genes lie upstream of the dorCDA structural genes. Recently, Kaplan and co-workers [26] have reported the presence of three orfs in R. sphaeroides that lie 5′ to dorR and have a role in the expression of DMSO reductase. It will be interesting to determine whether expression of any of these orfs is dependent on molybdate and MopB.

Acknowledgements

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

This work was supported by a grant from the Australian Research Council to A.G.M. and G.R.H. and an Australian Postgraduate Award to A.L.S. We thank Nigel Mouncey and Sam Kaplan helpful discussions. We thank Dr Conrado Moreno-Vivian for providing the nap gene cluster used in this study.

References

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