Abstract
Methanococcus voltae harbors genetic information for two pairs of homologous [NiFe]-hydrogenases. Two of the enzymes contain selenocysteine, while the other two gene groups encode apparent isoenzymes that carry cysteinyl residues in the homologous positions. The genes coding for the selenium-free enzymes, frc and vhc, are expressed only under selenium limitation. They are transcribed out of a common intergenic region. A series of deletions made in the intergenic region localized a common negative regulatory element for the vhc and frc promoters as well as two activator elements that are specific for each of the two transcription units. Repeated sequences, partially overlapping the frc promoter, were also detected. Mutations in these repeated heptanucleotide sequences led to a weak induction of a reporter gene under the control of the frc promoters in the presence of selenium. This result suggests that the heptamer repeats contribute to the negative regulation of the frc transcription unit.
IT was recognized early that RNA polymerases from archaea are much more complex than homologous enzymes from the other group of prokaryotes, bacteria (Zilliget al. 1989). This observation raised interest in the analysis of promoter structures in Archaea. Archaeal promoters resemble those of Eucarya and contain a TATA box and an initiator element (Thomm and Wich 1988; Reiteret al. 1990; Gohlet al. 1995). Indeed, two required transcription factors are homologues of the TATA-binding protein and TFIIB, constituents of the basic eucaryal transcription apparatus (for review see Thomm 1996). Given the particular mode of transcription initiation in archaeal cells, transcriptional regulation is also of interest. So far, only a few systems have been studied in some detail. In halophilic archaea, positive regulation by an activator protein governs the expression of gas vesicle genes (Röder and Pfeifer 1996; Krügeret al. 1998). Recently, a putative activator was described that is necessary for the transcription of the molybdenum formyl-methanofuran dehydrogenase in the methanogenic archaeon Methanobacterium thermoautotrophicum (Hochheimeret al. 1999). Negative regulation has been demonstrated for genes involved in nitrogen metabolism in Methanococcus maripaludis (Cohen-Kupiec et al. 1997, 1999). Classical repressors had earlier been described for lysogenic archaeal viruses (Ken and Hackett 1991; Stolt and Zillig 1992).
We have been studying the transcriptional regulation of genes encoding [NiFe]-hydrogenases in Methanococcus voltae. This archaeon harbors genetic information for four such enzymes (Halboth and Klein 1992), two of which contain selenocysteine residues as ligands of the Ni atom in their primary reaction sites. The other two have cysteinyl residues in the homologous positions. The two transcription units, vhc and frc, encoding the latter enzymes are transcribed only under selenium limitation (Berghöferet al. 1994). They are linked by an intergenic region containing all the cis-elements for the transcriptional regulation (Benekeet al. 1995). We were interested in determining what type(s) of regulation govern the transcription of the frc and vhc genes and whether or not the apparent coordinate regulation was due to common regulatory elements in the intergenic region. Our mutational analysis presented here suggests that both negative and positive regulation are involved and that an apparent silencer region mediates the coordinate regulation of the transcription of both gene groups.
MATERIALS AND METHODS
Strains and media: M. voltae PS, DSM 1537 was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Escherichia coli DH5α supE44ΔlacU169 (ϕ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 was obtained from Stratagene (La Jolla, CA). BW313 HfrKL16 PO/45 (lysA61-62) dut1 ung1 thi-1 relA1 (Kunkelet al. 1987) was a gift of H.-J. Fritz (Göttingen). The amino acid media used for M. voltae were described earlier (Berghöferet al. 1994; Sniezkoet al. 1998). Selective media for the isolation of transformants contained 5-10 μg/ml puromycin. E. coli was cultivated in LB medium, Terrific broth, 2YT medium (Sambrooket al. 1989), or Standard I (Merck, Darmstadt, Germany). Plates contained 1.5% agar. Selective media were supplemented with 100 μg/ml ampicillin or 50 μg/ml kanamycin.
Primers used for in vitro mutagenesis
Plasmids and primers: The plasmids used (Figure 1) were derived from Mip integration vectors (Gernhardtet al. 1990; Benekeet al. 1995; Sniezkoet al. 1998) into which the intergenic region (IR) between the vhc and frc gene groups was inserted and linked to the treA (Schöcket al. 1996) and/or uidA (Jeffersonet al. 1986) reporter genes (Benekeet al. 1995; Sniezkoet al. 1998) in between ClaI and NsiI sites. Plasmid MipvhcΔ was derived from Mipvhc. In this plasmid the intergenic region of Mipvhc was shortened by deleting the frc-proximal part of the intergenic region between the newly introduced EcoRI site (compare also Figure 2) and the tmcr terminator. For mutagenesis phagemid vectors pBluescript KS(+) (Shortet al. 1988) obtained from Stratagene and pSL1180 (Brosius 1989) purchased from Amersham Pharmacia Biotech (Braunschweig, Germany) were used. The primers used for in vitro mutagenesis are listed in Table 1.
DNA techniques: Standard techniques for plasmid preparation and cloning were those described in Sambrook et al. (1989) or Ausubel et al. (1996). In vitro mutagenesis followed the method of Kunkel et al. (1991). The method involves the introduction of uracil instead of thymine into DNA by an E. coli Dut- Ung- mutant. Upon transfection of this strain with a phagemid and helper phage, a uracil-containing single-stranded DNA of the phagemid template is produced. A primer carrying a mutation is then annealed to the template and the second strand is synthesized in vitro. The double-stranded, nicked phagemid is transformed into an E. coli Dut+ Ung+ strain. The repair system of the cell then recognizes uridyl residues and removes them. The strand with the mutation serves as template for the repair. Usually >95% of the analyzed clones carry the mutation. For the construction of mutants in the intergenic region the mutation was subcloned in phagemid vectors, mutagenized, and subsequently reinserted into the relevant plasmid after sequence verification.
Transformation: Transformation of E. coli was done by electroporation (Ausubelet al. 1996) using a Gene Pulser apparatus (Bio-Rad, Munich, Germany) at 2.5 kV, 25 μF, and 200 Ω. M. voltae was transformed employing liposomes (Metcalfet al. 1997; Sniezkoet al. 1998). Two micrograms of DNA per 109 cells was used. Single colonies were picked and cultivated in liquid medium.
Extract preparation and enzyme assays: The cell extracts used were centrifugation supernatants from cell lysates. They were prepared as described earlier (Benekeet al. 1995). Protein concentrations were determined using the dye binding assay (Bradford 1976) with bovine serum albumin as a standard. The conditions of the β-glucuronidase and trehalase tests were previously described (Benekeet al. 1995; Sniezkoet al. 1998). One unit of enzyme is defined as the activity leading to the hydrolysis of 1 μmol substrate per minute at 30°.
RESULTS
Coordinate regulation of the vhc and frc promoters: The transcription of the two gene groups frc and vhc, both encoding selenium-free [NiFe]-hydrogenases in M. voltae, is coordinately regulated. The gene expression increases after selenium deprivation. The gene groups are connected by an IR that contains the TATA-box-initiator-type promoters and Shine-Dalgarno sequences (see Figure 2). It was previously shown that the cis-elements for transcriptional regulation are contained in the intergenic region (Benekeet al. 1995). However, in these experiments the coordinate regulation of the frc and vhc promoters was not directly shown. In the meantime we have found that a second reporter gene, treA from Bacillus subtilis can also be expressed in M. voltae (Sniezkoet al. 1998). Therefore, the coordinate regulation of both promoters was demonstrated using cells transformed with a construct in which the frc promoter was linked to the uidA gene and the treA gene was connected to the vhc promoter (Miptu, Figure 1). Both activities were then determined in the same cell extracts. The results are shown in Table 2. Both reporter genes had only low activity in the presence of selenium. Both activities increased upon selenium deprivation. Thus the regulation of the vhc and frc promoters was coordinate.
—All four plasmids are based on the Mip integration vectors (Gernhardtet al. 1990; Benekeet al. 1995; Sniezkoet al. 1998). The reporter genes uidA from E. coli or treA from B. subtilis were put under the control of the vhc-frc IR or part thereof. The promoters thus linked to the reporter genes are indicated in parentheses. Mipvhc corresponds to Mipuid-vhc (Benekeet al. 1995) and served for the preparation of the deletion of the proximal part of the intergenic region yielding MipvhcΔ. Mipfrc2 is a derivative of Mipuid-frc (Benekeet al. 1995) in which two restriction sites were abolished: EcoRI at 4840 bp and a NdeI site within the intergenic region (compare Figure 2). Construction of the backbone for Mipv and Miptu was described by Sniezko et al. (1998). The IR-uidA-tmcr cassette for Mipv and Miptu was taken from Mipfrc2 and appropriately mutated. The source for the treA gene was Miptre (Sniezkoet al. 1998). The direction of the genes is indicated by the pointed boxes. pac, puromycin transacetylase gene from Streptomyces alboniger (Lacalleet al. 1989) used as selection marker; pmcr, methyl CoM reductase promoter; tmcr, terminator of the methyl CoM reductase transcription unit from M. voltae. hisA′ and ′hisA are sections of the hisA gene (Cueet al. 1985) of M. voltae used as homologous integration sequences.
Tandem heptamer repeats overlapping the initiator are involved in negative regulation of the frc promoter: As reported previously (Sorgenfreiet al. 1997), three heptameric sequences (II-IV, Figure 2b) repeated in tandem overlap the frc promoter initiator. The heptamer can be defined either as TGACTAA or ATGACTA. Assuming the first sequence, a fourth copy of the sequence was detected further upstream in the section (I, nucleotides 298-304, Figure 2b). To assess the effect of these sequences on the frc promoter activity, we subjected the sequences to site-directed mutagenesis in a construct in which the uidA gene was linked to the frc promoter (Mipfrc2, Figure 1). Mutation of the three boxes overlapping the initiator resulted in a small increase of the promoter activity (Table 3). Mutation of the single TGACTAA sequence (heptamer I) did not influence the expression of uidA. Mutation of only heptamer II belonging to the triple heptamer repeat had a slight but significant effect. This might mean that the relevant heptameric sequence reads ATGACTA, because the three adjacent heptamers II, III, and IV can be read in this way if the frame is shifted by one position in the 5′ direction; the apparently irrelevant sequence of heptamer I would then change to TTGACTA (see Figure 2).
Activity of the vhc and frc promoters dependent on selenium supply as simultaneously determined with the help of two reporter genes
—(a) Intergenic region between the vhc and frc gene groups of M. voltae. The wild-type sequence is available in the GenBank file under the accession no. X 61203. At the indicated positions 1-5 NdeI sites were introduced that were used to create deletions as described in the text. The changed hexanucleotide sequences are shown in italics and underlined. The position of a newly created EcoRI site used for the construction of plasmid Mip-vhcΔ (Figure 1) is shown with the same signature. It needed a G to C mutation at position 247. The TATA box and initiator (ATGA) elements of the two promoters are in boldface. The transcription initiation points (the G nucleotides within the initiator sequences) are marked with asterisks. The roman numerals I-IV indicate the positions of the four TGACTAA heptamers. (b) Mutational changes introduced into the heptamers. In the top row the wild-type sequences are shown. Note that the three adjacent heptamers will also look identical to each other if read in a frame shifted by one nucleotide in the 5′ direction. The second row indicates the changes introduced by site-directed mutagenesis in the mutants described in Table 3. The mutational changes are indicated in bold italics. The positions of the nucleotides in the intergenic region are also given by numbers. Alternative mutants were also tested and gave the same results.
Deletion of the region upstream of the frc gene group changes the regulatory pattern of the vhc promoter: We have suggested earlier (Sorgenfreiet al. 1997) that the heptamer sequences might be negative regulatory elements that influence the expression of both the frc and vhc promoters, acting as operators and silencers at the same time. This model predicted that removal of the heptamers would cause the induction of both the frc and vhc promoters in the presence of selenium. We therefore constructed an integration plasmid that carried the intergenic region linked to the uidA gene under the control of the vhc promoter and lacked the upstream region of the frc promoter including the heptamer repeats. While this construct exhibited vhc promoter activity (Table 4), the induction was incomplete. Thus, other regulatory cis-elements beside the heptamers probably influenced the vhc transcription. To identify those elements, we performed a deletion analysis of the intergenic region. Because the measurable trehalase activity in the cell extracts was comparatively low, the uidA reporter gene was used throughout in this approach. In an additional construct (Mipv, Figure 1) the intergenic region was therefore linked to the reporter gene so that the β-glucuronidase expression was governed by the vhc promoter.
Role of heptamer repeats on the regulation of the frc promoter
uidA expression under control of the vhc promoter of the intact vhc-frc-intergenic region or a part lacking the frc promoter-operator region
Deletion analysis of the intergenic region leads to the identification of further positive and negative regulatory elements: To perform deletion analyses, we introduced pairs of evenly spaced NdeI sites into the IR sequence to allow the deletion of defined parts of the intergenic region (Figure 2a). With the intact intergenic region, the expression of the uidA reporter gene was turned off in the presence of selenium when attached to either the frc or the vhc promoter. Deletion of a vhc-proximal part of the intergenic region (Δ1-3) led to a loss of function of the vhc promoter but, surprisingly, also strongly affected the frc promoter. Deletion Δ2-3 partially relieved the negative effect of selenium on both the frc and vhc promoters. However, it also led to reduction of the vhc promoter activity in the absence of selenium without affecting the frc promoter activity under this condition (compare lines 3 of Tables 5 and 6). This indicated the existence of positive regulation of the vhc promoter by an element contained in the region between the NdeI sites 2 and 3. This conclusion was confirmed by the Δ2-4 construct. This deletion also affected the activity of the frc promoter, which was reduced (Table 5, line 4). Similarly, deletions Δ3-4 and Δ3-5 both reduced uidA expression from the frc promoter. However, these deletions did not affect expression from the vhc promoter because the pattern of expression was similar to that observed with the complete intergenic region (compare Tables 5 and 6, lines 6). Upon deletion of region 1-5, both promoters were affected in the same way as with the Δ1-3 deletion.
Deletion analysis of the vhc-frc-intergenic region linked to the reporter gene uidA in frc direction
DISCUSSION
Positive and negative regulatory cis-elements of transcriptional regulation are known to be involved in regulation of both bacterial and eucaryal genes. The classical elements of bacterial negative transcriptional regulation are the operators located close to or overlapping with the promoter sequence as first described for the lac operon of E. coli (for review see Beckwith and Zipser 1970). They are binding sites for repressor proteins that interfere with the binding of the RNA polymerase or its action and therefore with the initiation of transcription. This type of regulation is also found in eukaryotes (for reviews see Renkawitz 1990; Hanna-Rose and Hansen 1996). Positive regulator elements can be either adjacent to the promoter as activator binding sites, like the classical Crp binding site (for review see Reznikoff 1992), or further upstream, like the NtrC binding site (Reitzer and Magasanik 1986) in E. coli. In the latter case they are functional counterparts of eucaryal enhancers. Silencers are regulatory elements that lead to a reduction of promoter activity at a distance. They are common regulatory elements in Eucarya (for review see Ogbourne and Antalis 1998). So far they have been found in only a few cases in Bacteria (Fletcher and Csonka 1995; Jubeteet al. 1995; Schnetz 1995; Schnetz and Wang 1996; Murphreeet al. 1997). These silencer regions can extend over more than 50 bp.
Deletion analysis of the vhc-frc intergenic region linked to the uidA reporter gene attached to the vhc promoter
—Model describing the coordinate regulation of the gene groups encoding selenium-free hydrogenases in M. voltae. The gene groups are indicated as large pointed boxes. They point in the direction of their transcription. The promoters are shown as smaller pointed boxes proximal to the genes. Negative regulatory elements are shown in black; positive regulatory elements are shown as open boxes. The locations of the NdeI sites introduced for the deletion mutagenesis are numbered as in Figure 2. Note that the relative positions of the silencer element and the positive regulatory element shown within section 2-3 are arbitrary. They could be overlapping.
We were interested in understanding the coordinate regulation of the two transcription units encoding selenium-free [NiFe]-hydrogenases in the methanogenic archaeon M. voltae. Our experiments have revealed at least four regulatory regions in the intergenic region linking the two transcription units frc and vhc. They are depicted in Figure 3. The repeated heptamer overlapping with the initiator of the frc promoter resembles an operator. However, it plays only a minor role. It does not influence the vhc promoter, which was found to be inactive in the presence of selenium in a construct in which the tandem repeats were deleted (data not shown). The main negative regulatory region is located in region 2-3 (Figures 2 and 3), because its deletion can lead to roughly 40-fold induction of both promoters. It is therefore a common regulatory element. Still, further mutational analysis is needed to rule out that this interval contains two very closely neighboring independent promoter-specific elements. In any case, the element(s) would function at a distance of at least 100 bp, which is common for silencers and corresponds to the distances seen with bacterial enhancers and silencers or upstream regulatory sequences in eucaryal promoter regions.
As mentioned, negative regulation by silencers is rare in bacteria. In one of the described cases elements on both sides of the affected promoter are needed (Schnetz 1995). This is ruled out in our case because the affected promoters drive reporter genes that are followed by plasmid sequences. It is highly unlikely that a putative silencer protein would specifically interact with elements in these sequences. In contrast to the silencer region, the two positive regulation elements that we have detected in regions 2-3 and 3-4 are specific for the vhc or frc promoters, respectively. The coordinate regulation therefore appears to rely mainly on the silencer.
The results obtained with deletions Δ1-3 and Δ1-5 show that both lead to a strong reduction or complete loss of the vhc or frc promoter activity, respectively. We have found that a Δ1-2 deletion has the same effect on the frc promoter (data not shown). These results are not incorporated in the model shown in Figure 3. In principle, the 1-3 region could contain another activating element for the vhc promoter. However, the simultaneous negative effects of deletions Δ1-2, Δ1-3, and especially Δ1-5 on both promoters are difficult to understand, because in the latter case the silencing element located in section 2-3 is removed. Further investigations will therefore be needed to explain these findings that could be due to a more general effect such as a change in DNA or even chromatin structure, which could also influence the promoter activities as reported for the known bacterial silencers (Schnetz and Wang 1996).
Acknowledgments
The excellent technical assistance of Danny Stingel is gratefully acknowledged. We thank Ken Jarrell for critically reading the manuscript and Hannelore Steinebach for help with its preparation. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 395) and Fonds der Chemischen Industrie. I. Noll is a fellow of the Gottlieb Daimler- and Karl Benz-Stiftung.
Footnotes
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Communicating editor: W. B. Whitman
- Received March 5, 1999.
- Accepted April 26, 1999.
- Copyright © 1999 by the Genetics Society of America
