Institut für Mikrobiologie und Genetik, Technische Universität Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany
Correspondence
Felicitas Pfeifer
pfeifer{at}bio.tu-darmstadt.de
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ABSTRACT |
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Present address: Klinische Pharmakologie, Klinikum der Universität Frankfurt, Theodor Stern Kai 7, D-60590 Frankfurt, Germany.
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INTRODUCTION |
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The basal transcription machinery of archaea appears to be a simpler version of the eukaryotic transcription system (Bell & Jackson, 1998). A 12-subunit RNA polymerase, the TATA-box binding protein TBP, and the transcription factor TFB constitute the archaeal preinitiation complex. The TATA box is a highly conserved 8 bp sequence located 2428 nt upstream of the transcription start site. An analysis of this sequence element in hyperthermophilic archaea indicates that it constitutes the TBP-binding site, especially when TBP is complexed with TFB (Kosa et al., 1997
; Littlefield et al., 1999
). The TFB-responsive element BRE, a purine-rich region of 7 bp with the consensus sequence cRnaAnt, is located upstream and adjacent to the TATA box (Qureshi & Jackson, 1998
; Bell et al., 1999a
; Littlefield et al., 1999
). BRE defines the orientation of the preinitiation complex, and is also important for promoter strength in hyperthermophilic archaea. Structural data obtained with the ternary transcription initiation complex of Pyrococcus woesei indicate that TFB contacts TBP and DNA sequences up and downstream of the TATA box (Kosa et al., 1997
; Littlefield et al., 1999
). Additional proteins involved in transcription are TFE
and the cleavage induction factor TFS (Bell et al., 1999b
, 2001
; Hausner et al., 2000
; Hanzelka et al., 2001
). In vitro analyses indicate that TFE has a stimulatory function and stabilizes the interaction between TBP and the TATA box when promoter recognition is not optimal.
In vitro transcription systems are available for methanogenic and hyperthermophilic archaea, and have been used to study the formation of the preinitiation complex and the action of various transcription regulators (Hochheimer et al., 1999; Bell et al., 1999a
; Brinkman et al., 2000
; Enoru-Eta et al., 2000
; Leonard et al., 2001
; Ouhammouch et al., 2003
). However, an in vitro transcription system is not available for halophilic archaea. The majority of the studies on the transcriptional activator protein GvpE have been done in vivo. It is not yet known just how GvpE activates transcription at the pA promoter, and whether it activates other p-vac promoters besides pA.
A second regulatory protein, GvpD, appears to be involved in the repression of gas vesicle formation, at least in the case of Haloferax mediterranei (Englert et al., 1992b; Pfeifer et al., 1994
, 2001
). Studies on the mc-vac region involved in the gas-vesicle formation of Hfx. mediterranei indicate an overproduction of gas vesicles in
D transformants lacking GvpD, which can be reduced to wild-type level in
D/Dex transformants (Englert et al., 1992b
; Pfeifer et al., 1994
). At the transcript level, the amount of mc-gvpA mRNA is similar in A
DE (lacking GvpD) and ADE transformants, but comparison of
-galactosidase activities found in the respective mcA-bgaH-
DE and mcA-bgaH-DE transformants shows a reduction of the mcA-promoter activity in the presence of GvpD (Zimmermann & Pfeifer, 2003
). It is possible that the high stability of the mc-gvpA mRNA, with a half-life of 160 min in Hfx. volcanii (Jäger et al., 2002
), is the reason for these differences. The amino acid sequence of GvpD indicates a conserved p-loop motif near the N-terminus typical of ATP/GTP binding proteins and important for the repressor function of GvpD (Pfeifer et al., 2001
). The GvpD and GvpE proteins of Hfx. mediterranei are able to interact in vitro, and it is likely that this also occurs in vivo (Zimmermann & Pfeifer, 2003
). The GvpD protein encoded by the p-vac region might have a similar function in the repression of gas-vesicle formation.
In this report, we investigated the regulation of the four promoters of the p-vac region in Hfx. volcanii transformants. The basal promoter activities were determined and compared to the promoter activites found in the presence of GvpE and/or GvpD. The results indicated that GvpE activated the pD promoter in addition to pA, and that the pA-bgaH construct showed less activity when GvpD was present in addition to GvpE. In contrast, neither the pF nor the pO promoters were affected. We also analysed the pA promoter by a 4 nt scanning mutagenesis within a 50 nt region to determine the sequences important for basal and GvpE-induced transcription in vivo.
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METHODS |
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The construction of pD-bgaH, pF-bgaH and pO-bgaH involved the amplification of the respective promoter fragment [pD: 161 bp XbaINcoI fragment, positions 767 of X64729 plus 1100 of X55648, primer pair p-5'D-XbaI and leaderp-D-NcoI (5'-GGTGAGCCATGGTGGGTGAACTCAT-3'); pF: 275 bp XbaINcoI fragment, position 20072281 of X55648, primer pair pF-XbaI (5'-AGAACTGCTCTAGAATCTCCGGCGGCTG-3') and leader-p-F-NcoI (5'-GATACCGTATGCCATGGGGTTCTCAGTCATTGG-3'); pO: 84 bp XbaINcoI fragment, position 25912674 of X64729, primer pair pO-XbaI (5'-GCGCGAAGATCTAGAATCCGCGATCG-3') and leader-p-O-NcoI (5'-CAGATCGATCCATGGCTGGATCTGCCATG-3')]. These promoter fragments were fused with the 2203 bp NcoIBamHI fragment, encompassing the bgaH reading frame [position 23624564 of U70664; primers bgaH-NcoI (5'-CATTGTCCATGGCAGTTGGTGTCTG-3') and bgaH-BamHI (5'-GTGACGCGGATCCGCGTGTGTAC-3')] and inserted in pBSIISK+. After DNA sequence determination of the promoterbgaH fusion region, the respective fragments encompasssing pX-bgaH were transferred as XbaIBamHI fragments to pWL102. Both vectors used in this study (pWL102 and pJAS35) are low-copy-number plasmids with <10 copies per cell. The Genbank accession numbers of the p-vac subfragments are given in the footnote.
Constructs used for the scanning mutagenesis.
The mutant pA promoters were constructed by recombinant PCR, fused to the bgaH reading frame and inserted into pWL102. The desired 4 nt mutations were introduced using pA-bgaH in pBSIISK+ as template. The oligonucleotides used for this procedure are summarized in Table 1. Two PCR reactions were performed to amplify overlapping subfragments harbouring the mutations in the overlap. The first PCR was carried out with the primers 01b through 09b, in combination with pA-XbaI. In the case of primers N1b through N4b, the primer M13 reverse (complementary to a pBSK sequence) was used in order to obtain large enough fragment sizes for the gel extraction. The second series of PCR was performed with oligonucleotides 01a through 09a, and N1a through N4a, together with primer bgaH-extra (5'-TATCGGTCGGTCAGCACG-3'). Each of the amplified fragments was purified by gel electrophoresis and eluted using the Ultrafree-DA system (Millipore). The two overlapping subfragments were used as templates together with the primers pA-XbaI and bgaH-extra for the third PCR amplification. In each case, a 512 bp fusion fragment containing the mutated pA promoter was obtained and subsequently fused to the 5'-terminal part of bgaH. The fragments were purified by gel electrophoresis and eluted using the QIAquick gel extraction kit (Qiagen). The various 120 bp pA mutant promoter fragments were excised from these constructs as XbaINcoI fragments and were used to substitute the 120 bp wild-type pA promoter in pA-bgaH in vector pBSIISK+. The correct mutation and the fusion of the pA promoter to bgaH were determined by DNA-sequence analysis in each case. The various 2·3 kb pA-bgaH fragments were isolated as XbaIBamHI fragments and inserted in pWL102.
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Isolation of RNA and transcript analysis.
RNA was isolated from Hfx. volcanii transformants according to the method of Chomczynski & Sacchi (1987). RNA produced during the exponential growth phase was isolated from cultures at OD600 0·20·4, whereas RNA from stationary-phase cells was isolated at OD600
2. Northern analyses involved electrophoresis of 5 or 10 µg RNA on denaturing formaldehyde-containing 1·2 % (w/v) agarose gels, followed by transfer to nylon membranes (Ausubel et al., 1988
). A strand-specific RNA probe was synthesized, using the respective p-gvp gene (A, D, F or O) cloned in pBluescript, and used as template for the T3/T7 polymerase. The RNA was labelled using the DIG RNA labelling kit (Roche). Northern hybridization was carried out as described by Ausubel et al. (1988)
, but the hybridization solution contained 10 % (w/v) dextran sulfate (Sigma), 1 % (w/v) SDS, and 0·5 % (w/v) skimmed milk powder.
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RESULTS |
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Northern analyses were done with RNA samples derived from the exponential and stationary growth phases of these transformants, and blots were hybridized with the respective gvp gene probe (Fig. 2). An example of a methylene-blue stained agarose gel with separated total RNA is shown in Fig. 2(a)
, which demonstrates the quality and relative amounts of 23S and 16S rRNA in these experiments. This particular blot was used for hybridization with the p-gvpA probe shown in Fig. 2(b)
. Relatively minor amounts of the 0·27 kb p-gvpA mRNA were observed with the p-gvpA transformant (Fig. 2b
). Minor amounts of p-gvpA mRNA were also seen in the A/Dex transformant, implying that GvpD was not repressing the pA promoter. In both transformants, slightly larger amounts of gvpA mRNA were observed in the samples derived from exponential growth. The A/DEex and A/Eex transformants contained significantly larger amounts of the p-gvpA mRNA in the exponential growth phase, demonstrating that GvpE activated the pA promoter (Fig. 2b
). The large amounts of transcripts in these samples were due to the early and high expression of GvpE under fdx-promoter control in pJAS35. Similar amounts of p-gvpA mRNA were observed in the A/DEex and A/Eex transformants, suggesting that GvpD did not reduce the GvpE-mediated stimulation of the pA promoter. The O transformant produced the 0·4 kb p-gvpO mRNA predominantly in the stationary growth phase, and no significant alterations were seen in the O/Dex and O/Eex transformants, suggesting that the pO promoter was influenced neither by GvpD nor by GvpE (Fig. 2c
).
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Further analyses were carried out with leadD transformants harbouring a construct containing a 190 bp fragment encompassing the pD promoter plus DNA encoding the 71 nt p-gvpD mRNA leader (see Fig. 1). The transcription of this fragment resulted in a 0·24 kb mRNA (Fig. 2e
). Minor amounts of this transcript were seen in the leadD transformant, indicating the low basal activity of the pD promoter. The Dex transformant (containing the p-gvpD reading frame expressed in pJAS35) lacked this 0·24 kb transcript due to the absence of the sequence encoding the mRNA leader in this construct. Similar amounts of the 0·24 kb RNA were seen in leadD and leadD/Dex transformants, implying that GvpD did not repress the pD promoter. Large amounts of the 0·24 kb transcript were seen in the leadD/DEex and leadD/Eex transformants, which demonstrated that GvpE was able to activate the pD promoter (Fig. 2e
). Again, the transcription was high during exponential growth, due to the early expression of p-gvpE or p-gvpDE under fdx promoter control in pJAS35. No significant reduction was seen in the leadD/DEex compared to leadD/Eex transformant, suggesting that the presence of GvpD did not reduce the GvpE-mediated activation of the pD promoter.
In the case of the F transformants, Northern analysis showed the presence of a 1·2 kb p-gvpF mRNA (including one larger and several smaller transcripts), predominantly in the exponential growth phase (Fig. 2f). This time point of expression was in agreement with the appearance of the gvpFGHIJKLM mRNA in Hbt. salinarum pHH1 (Offner & Pfeifer, 1995
). Since the amount of the gvpF-specific mRNAs was not significantly altered in the F/leadDex (used to determine a putative effect of the p-gvpD mRNA leader), F/Dex and F/Eex transformants, and only slightly enhanced in the F/DEex transformants, these results suggested that the pF promoter was neither affected by GvpD nor by GvpE.
Determination of the p-vac promoter activities using bgaH as reporter
The four promoters of the p-vac region were also fused with the bgaH reading frame to determine the specific -galactosidase activities in the respective transformants. Each promoter fragment used encompassed the TATA box and upstream sequence, the transcription start site, and the region encoding the respective mRNA leader (5'-UTR) of the respective gvp gene. The p-gvpA mRNA contains a 21 nt 5'-UTR, the p-gvpD mRNA a 71 nt leader, and p-gvpF a 169 nt 5'-UTR (DasSarma et al., 1987
; Jones et al., 1989
; Offner & Pfeifer, 1995
). In contrast, the p-gvpO mRNA starts only 1 nt upstream of the AUG initiation codon, and does not contain an mRNA leader (Offner et al., 1996
). The mRNA leader regions of p-gvpA, p-gvpD and p-gvpF were included, since it was not known whether and where GvpE (and GvpD) could possibly bind. However, these 5'-UTRs may also influence the stability of the transcript and its translation.
The resulting pA-bgaH, pD-bgaH, pF-bgaH and pO-bgaH constructs were used to transform Hfx. volcanii, and the colonies formed were sprayed with X-Gal for an initial inspection of -galactosidase activity. Colonies of the pA-bgaH and pF-bgaH transformants turned dark blue, and colonies of the pD-bgaH transformants were light blue (data not shown). In contrast, those of the pO-bgaH transformants remained red. The ONPG assay was used to determine the specific
-galactosidase activities of the various transformants (Table 2
). The pA-bgaH transformant yielded a mean specific
-galactosidase activity of 21 mU mg1 in stationary growth, and slightly reduced activities were seen in the pA-bgaH/Dex transformant (Table 2
). Much higher
-galactosidase activities (25-fold enhanced) were observed with the pA-bgaH/Eex transformant, whereas the pA-bgaH/DEex transformant showed lower amounts (9-fold enhanced), suggesting that the presence of GvpD reduced the GvpE-mediated activation (Table 2
). Together, these results underlined the function of GvpE as transcriptional activator at the pA promoter, but also implied a repressing function of GvpD in the presence of GvpE. The basal pD promoter activities determined for the pD-bgaH transformant were very low, but the pD-bgaH/Eex transformant yielded an almost 20-fold increased
-galactosidase activity, demonstrating the activation of the pD promoter by GvpE (Table 2
). The basal promoter activity of pF-bgaH was higher than that of pD-bgaH, and not significantly altered in the presence of Dex, Eex or DEex. These findings emphasize the fact that the pF promoter was not affected by these regulatory proteins (Table 2
). Very minor amounts of
-galactosidase activity were determined for the various pO-bgaH transformants, despite the fact that significant amounts of bgaH mRNA were produced in each pO-bgaH transformant (data not shown). These results suggested that the pO-bgaH mRNA was not very well translated. In summary, the data indicated that GvpE acts as transcriptional activator at the pA and pD promoters, but also suggested that the GvpE-mediated activation of pA was reduced in the presence of GvpD. In contrast, the pF promoter was affected neither by GvpD nor by GvpE.
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With respect to the GvpE-mediated activation, minor activations were observed with the pA-bgaH mutants C, D and (to a lesser extent) K (Fig. 4b). The smallest
-galactosidase activities were observed with mutants C and D, carrying alterations upstream of the BRE sequence element. Mutant C (region 40 to 42 altered) was not affected in basal pA promoter activity, whereas mutant D yielded a dramatic reduction in both basal and GvpE-induced pA-promoter activity (Fig. 4
). From these results it appeared that the region upstream and adjacent to BRE was most important for GvpE-mediated activation. Mutants A and B (carrying mutations further upstream), and mutants H, I, J, L and M (with mutations closer to the transcription start site), exhibited
-galactosidase activities similar to those observed for the GvpE-induced pA promoter of the wild-type (Fig. 4
).
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DISCUSSION |
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A reduction of the GvpE-induced pA promoter activity by GvpD could not be observed at the level of the p-gvpA transcript in p-gvpA/DEex transformants. However, the respective pA-bgaH/DEex transformants demonstrated a role for GvpD in the repression of the pA promoter, since these transformants produced less -galactosidase activity than pA-bgaH/Eex transformants. Earlier results on the mc-vac region of Hfx. mediterranei have shown that transformants harbouring the subfragments ADE and A
DE do not show a reduction in the mc-gvpA mRNA (Zimmermann & Pfeifer, 2003
). However, the reducing effect of GvpD on GvpE-mediated mcA promoter activation can be observed in transformants harbouring the mcA-bgaH-DE (or mcA-bgaH-
DE) constructs (Zimmermann & Pfeifer, 2003
). It is possible that the very stable gvpA mRNAs are less useful for demonstrating the reduction of the GvpE-mediated activation of the A promoters in the presence of GvpD. The half-life of the mc-gvpA mRNA (as determined by Northern analysis after addition of actinomycin D) is, at 160 min, extremely long in Hfx. volcanii transformants (Jäger et al., 2002
). The half-life of the p-gvpA mRNA appears to be similar (unpublished data).
The combined results on the activity and regulation of the four p-vac promoters allowed the following explanation of the expression of p-vac in Hbt. salinarum. The early appearance of the p-gvpFGHIJKLM and p-gvpO mRNAs was due to the basal activities of the pF and pO promoters; GvpE did not induce an enhanced transcription. The reductions in the p-gvpFGHIJKLM and p-gvpO mRNAs in the stationary growth phase were not caused by GvpD, since this protein has no influence on the pF and pO promoter activities. The reduced amounts of these mRNAs were presumably due to an overall reduced transcription initiation in the stationary growth phase, combined with the degradation of the transcripts. In contrast, the increased amounts of the p-gvpDE transcript in the stationary growth phase could be assigned to the autoregulation of the pD promoter by GvpE. The activation of the pD promoter and formation of the p-gvpDE mRNA led to a larger production of GvpE, but also to a higher amount of GvpD in the growth phase. In the case of the mc-vac region, a small amount of GvpE is already sufficient for maximal mcA promoter activity (Zimmermann & Pfeifer, 2003), and this could also be true for the p-vac region of Hbt. salinarum. The pA promoter is active throughout growth and drives the expression of the genes encoding the gas vesicle structural proteins GvpA and GvpC. During exponential growth, the basal pA promoter activity results in the formation of p-gvpA mRNA, and the appearance of GvpE in stationary growth (due to the expression of the p-gvpDE transcript) leads to a severalfold induction of pA in this growth phase. The production of GvpD presumably reduces the action of GvpE, since lower
-galactosidase activity was observed in the pA-bgaH/DEex transformants compared to pA-bgaH/Eex transformants. It should be stressed that, although both regulatory proteins are encoded by consecutive genes and cotranscribed, the activation by GvpE exceeds the repressing function of GvpD. Results obtained with these Hfx. mediterranei regulatory proteins indicate that small amounts of mcGvpE are sufficient for a high activity of the mcA promoter (Zimmermann & Pfeifer, 2003
). Thus, GvpE is either highly active and/or binds with high affinity to the promoter region (or to subunits of the basal transcription machinery).
Efforts to determine the promoter region required for the GvpE-mediated activation
The DNA sequences involved in the GvpE-mediated activation of the pA promoter have not yet been determined. In vitro analyses demonstrate that GvpE is able to dimerize in solution (Plößer & Pfeifer, 2002). However, all attempts to study the DNA-binding properties of GvpE in vitro have so far failed, mainly due to the requirement of this halophilic protein for 2 M salt. In this report, we tried to identify the region most important in vivo for GvpE-mediated activation in the pA promoter. Scanning mutagenesis was carried out with a 50 nt region located upstream of the transcription start site of the p-gvpA gene. Alteration of sequences found upstream or within the BRE sequence element (mutants D, F), overlapping the TATA box (mutant G), and around position 10 (mutants K, L), reduced the basal transcription to almost zero (<0·5 mU mg1
-galactosidase activity), and also influenced the GvpE-induced activity of this promoter. Except for the region around position 10, all of these DNA sequences are involved in the recruitment and binding of TBP and TFB in hyperthermophilic archaea, as demonstrated by footprint analyses (see Fig. 3
) (Qureshi & Jackson, 1998
; Littlefield et al., 1999
). Thus, the in vivo analyses presented here confirmed that these promoter sequences are also important in halophilic archaea, since mutations strongly influence basal transcription. Some of the mutations between the TATA box and the transcription start site led to an increase of the GC content of this sequence. Most likely, these additional GC base pairings raised some conflicts with the open complex formation, resulting in the reduced
-galactosidase activity observed in these mutants. An almost 40-fold increase of transcription was seen with mutant E, which contains the sequence CGGTATC instead of ACACATC as putative BRE element sequence in the pA promoter. This sequence exhibits a higher degree of conservation with respect to the consensus BRE element sequence cRnaAnt (with R=A/G, and n=any nucleotide) determined for hyperthermophilic archaea (Qureshi & Jackson, 1998
; Littlefield et al., 1999
). The strong increase in
-galactosidase activity in this mutant suggests that the BRE element sequence makes a major contribution to (or even determines) the strength of the pA promoter. Hbt. salinarum and Hfx. volcanii contain multiple TFB proteins that might be involved in gene regulation (Baliga et al., 2000
). Since the basal promoter activity of mutant E was already high during exponential growth it is also possible that a different TFB protein recognized this novel BRE element, resulting in the enhanced transcription observed.
With respect to the GvpE-mediated activation, some of the mutants with almost undetectable basal transcription (mutants G, H, I and L) were induced by GvpE to a normal extent, whereas others exhibited reduced activation (mutants F, J and K). Mutant D lacked GvpE-mediated activation entirely. It is difficult to decide whether a reduced binding of the basal transcription machinery was responsible for these effects, or whether the sequences altered were also involved in the GvpE-interaction. However, mutant C, which harbours a DNA sequence alteration upstream of the BRE element, showed a normal amount of basal transcription, yet the GvpE-mediated activity was strongly reduced (<20 % of wild-type). Mutations further upstream had no effect on basal or GvpE-induced promoter activation. The sequence AACCA, altered in mutant C, could be the GvpE binding site. However, this sequence is not conserved in the promoters mcA, cA and mcD that are also stimulated by GvpE. Further analyses are required to determine the GvpE-binding site unambiguously. Overall, the results obtained here imply a close contact of GvpE with the core transcription machinery. GvpE can promote or stabilize the binding of TBP or TFB, in a similar way to the transcriptional activator protein Ptr2 of Methanococcus jannaschii (Ouhammouch et al., 2003).
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ACKNOWLEDGEMENTS |
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Received 4 February 2004;
revised 16 March 2004;
accepted 22 March 2004.
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