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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INTRODUCTION |
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The formation of gas vesicles requires the expression of the 14 genes gvpACNO and gvpDEFGHIJKLM, which are located in two opposite clusters in the vac region (Horne et al., 1991; Englert et al., 1992a
). The TATA boxes of the promoters of gvpA (PA) and gvpD (PD) are separated by 50 bp in the plasmid-borne p-vac region of Hbt. salinarum PHH1 and the mc-vac region of Hfx. mediterranei. PA and PD are both activated by the endogenous activator protein GvpE (Röder & Pfeifer, 1996
; Zimmermann & Pfeifer, 2003
; Hofacker et al., 2004
). Hfx. mediterranei harbours the single mc-vac region, whereas Hbt. salinarum PHH1 contains, in addition to p-vac, the related but distinct c-vac region. Both vac regions are similar but not identical to the gvp1 (p-vac) and gvp2 (c-vac) gene clusters of Halobacterium sp. NRC-1 (Ng et al., 2000
). The expression of gvpACNO leads to the synthesis of the gas vesicle structural proteins GvpA and GvpC. The second operon encodes (among other proteins presumably involved in gas vesicle assembly, and minor gas vesicle structural proteins; Shukla & DasSarma, 2004
) the two regulatory proteins GvpD and GvpE. Four promoters (PpA, PpD, PpF and PpO) drive the expression of p-vac, leading to the constitutive production of spindle-shaped gas vesicles in Hbt. salinarum PHH1 (Offner et al., 1996
; Hofacker et al., 2004
). A promoter scanning mutagenesis performed on a 50 bp region upstream of the transcriptional start site of PpA determined that the sequences of BRE and the TATA box, as well as a sequence around position 10, influence the basal transcription. Furthermore, an adaptation of the putative BRE sequence element to the archaeal consensus BRE element sequence results in a significantly enhanced basal PpA promoter activity. These analyses also imply that the sequence AACCA located upstream and adjacent to BRE is involved in the GvpE-mediated activation, suggesting a close contact of GvpE with the core transcription machinery (Hofacker et al., 2004
).
The second vac region of Hbt. salinarum PHH1, c-vac, is only partly expressed in this wild-type strain (due to the minor activity of PcD), but the c-gvpACNO genes are not expressed at all (Pfeifer et al., 1997). Gas vesicles due to c-vac are only formed in the p-vac deletion mutant Hbt. salinarum PHH4 (Krüger & Pfeifer, 1996
). Using the gvp negative Hfx. volcanii as recipient strain, earlier investigations showed that a basal promoter activity of PcA is not detectable and that this promoter is only active in the presence of cGvpE in c-gvpA/cEex transformants that contain the c-gvpE reading frame expressed under the control of Pfdx in pJAS35 in addition to c-gvpA (Krüger et al., 1998
). Similar results on the PcA activity have been obtained using the bgaH reading frame encoding an enzyme with
-galactosidase activity as reporter (Holmes & Dyall-Smith, 2000
; Gregor & Pfeifer, 2001
). Again, in contrast to PcA, the PpA and PmcA promoters yield basal promoter activities that are significantly enhanced in the presence of the respective homologous GvpE proteins. In contrast to PcA, these two PA promoters are also activated by heterologous GvpE proteins. GvpE resembles eukaryotic basic leucine-zipper (bZIP) proteins and is able to dimerize in solution (Krüger et al., 1998
; Plößer & Pfeifer, 2002
). More recent analyses on mcGvpE and the second regulatory protein, mcGvpD, involved in the repression of gas vesicle formation of Hfx. mediterranei, show that GvpE and GvpD are able to interact (Zimmermann & Pfeifer, 2003
).
In the present study, using the bgaH reporter system, we compared the activities of the three gvpA promoters to that of the strong and constitutive Pfdx promoter of the fdx gene encoding the (2Fe2S) ferredoxin of Hbt. salinarum (Pfeifer et al., 1993) in Hfx. volcanii transformants. In addition, the activity of the PbgaH promoter was determined. The PbgaH-bgaH and Pfdx-bgaH transformants yielded large amounts of BgaH activities, whereas the basal PpA and PmcA promoter activities were rather low. Again, a basal PcA activity was not detectable in PcA-bgaH transformants. To investigate whether the PcA-TATA box and BRE were the reason for the latter observation, promoter chimeras were constructed between PcA and PpA and analysed for reporter gene expression. None of these PcA-pA promoter variants yielded a detectable basal promoter activity, but the activity was enhanced in the presence of GvpE when the BRE element and/or the TATA box were exchanged. In addition, all three GvpE proteins were able to activate the PcA-pA promoter variants that contained the PpA-BRE element (plus at least 5 nt further upstream), whereas the original PcA promoter and the derivatives still containing PcA-BRE were only activated by cGvpE. To determine the sequences important for the GvpE-mediated activation, a 4 bp scanning mutagenesis was done with the PmcDPmcA region separating the BRE elements of the two mc-vac promoters. The results obtained suggested that two conserved regions adjacent to BRE were involved in the GvpE-mediated activation of PmcA.
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METHODS |
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The BRE and TATA substitutions in PcA were introduced by recombinant PCR using complementary primers including the mutations, and the c-gvpA gene in pBSK+ as template. Two PCR reactions were performed to amplify two subfragments harbouring the inserted mutations in the overlapping end. The cA-pA-TATA, cA-pA-BRE and cA-pA-BRETATA fragments were amplified using the mutation primer BT-null (CGGACACTCCCTGTAGTT) plus cA-NcoI (Gregor & Pfeifer, 2001) for the first PCR, and the primers TATA (AGGGAGTGTCCGCATAAGCGCCGTTGTGA), BRE (AGGGAGTGTCCGGAAAACGATGTGTGTGAGTTCAA), or BRETATA (AGGGAGTGTCCGCATAAGGATGTGTGTGAGTTCAA) plus cA-XbaI for the second PCR. The full-size fragments were finally amplified using the cA-XbaI and cA-NcoI primers (Gregor & Pfeifer, 2001
).
The PpA-cA promoter variants were constructed in the following way. The production of the promoter fragment +5u involved primer pair cA-NcoI plus pA-cA1 (ACTGGTGAAACCATACACATCGTT), and the pA-cA promoter (Gregor & Pfeifer, 2001) as template. The second PCR was done with the mutation primer pA-cA2 (ATGGTTTCACCAGTCGTTACGGCGCTCGTAA) and cA-XbaI. A third PCR with both amplicons as template and primers cA-XbaI and cA-NcoI yielded +5u. Similarly, the +10u promoter was constructed using mutation primer pA-cA-3 (ATGGTTTCACCAGTCGTTATGTCTCTCGTAATAGTT). The promoter chimera 5u was amplified using c-gvpA as template and mutation primer pA-cA-4 (GTTTTCCGGACACTCCCTGTAGTT) plus cA-NcoI. In the second PCR, primer pair pA-cA-5 (TGTCCGGAAAACGATGTGTATGGTTTCAACCCCCTTT) plus cA-XbaI were used, and the final promoter fragment was amplified using the products of both PCRs as template and primers cA-XbaI and cA-NcoI. The promoter 10u was produced in a similar way, but involved the mutation primer pA-cA-6 (TGTCCGGAAAACGATGTGTATGGGTTCAACCCCCGTTT). For the construction of 5d, the mutation primer pA-cA-7 (CGTTTCGGCGCTCGTAATAGTTCGCT) was used together with cA-XbaI and c-gvpA as template; the second PCR involved primer pA-cA-8 (AGCGCCGAAACGACTGGTGAAACCACAACGGCGGTTTTCCGGACACT) together with cA-NcoI. The construction of 10d was similar, but involved the mutation primers pA-cA-8 (AGCGCCGAAACGACTGGTGAAACCACAACGGCGGTTTTCCGGACACT) and pA-cA-9 (AGCGCCGAAACGACTGGTGAAACCATACCGGCGGTTTTCCGGACACT).
The mcA promoter mutant mcA-Del was amplified using primer pair mcA-NcoI and mcA-Del-Pal (CCAAACTATCTAGATGTTTGACTCATTACGAGAGGTGAAACGGTTGCACCAACACGAATG). The promoter mutants mcA-M1 through mcA-M6 involved the substitution of 4 bp upstream of the TATA box. The first PCR product was obtained using primer mcA-M0 (Table 1) together with mcA-NcoI, and mc-gvpA as template. The second PCR involved primer mcA-M1 (or mcA-M2 through M6; Table 1
) together with mcA-XbaI. The mutants mcA-M7 through mcA-M11 were amplified using the respective oligonucleotides mcA-M7 through mcA-M11 together with mcA-NcoI and mc-gvpA as template. Each of these amplified fragments was purified by gel electrophoresis, and the PAmut promoter fragments were obtained by XbaI/NcoI digestion and used to substitute the wild-type PA promoter in the respective PA-bgaH fragment in vector pBSK+. In each case, the correct mutation and fusion of the mutant promoter to bgaH was determined by DNA sequence analysis. Each of these mutant PmcA-bgaH fragments was isolated as an XbaIBamHI fragment and inserted in pWL102 for transformation of Hfx. volcanii.
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RESULTS |
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Search for the sequence affecting the GvpE-mediated activation of PmcA
The PmcA promoter of Hfx. mediterranei offers the highest promoter activity of all PA promoters when induced with GvpE, and also yields a measurable basal promoter activity (Fig. 1; Gregor & Pfeifer, 2001
). A 4 bp scanning mutagenesis was performed with the 49 bp region separating the TATA boxes of PmcA and PmcD, and the resulting mutant PmcA-bgaH transformants were investigated for BgaH activities (Fig. 4
). None of these 4 bp alterations affected the basal activity of PmcA (Fig. 4b
). The analysis of the GvpE-mediated activation yielded reduced activities (1923 % of the wild-type activity) in mutants carrying the alterations adjacent to BRE (mcA-M1 through mcA-M3) and in the centre of this region (mcA-M5 through mcA-M7), where the most significant reductions (610 % of the wild-type activity) were observed (Fig. 4a
; the PmcA sequences affected are indicated in bold in Fig. 4b
). These analyses suggested that the sequence TGAACCAA close to BRE, and also the sequence TGAAACGG in the centre of the intergenic region, were important for the GvpE-mediated activation of PmcA. Mutant mcA-Del incurred a 3 bp deletion 6 bp upstream of BRE (Fig. 4b
). This deletion did not affect the basal promoter activity of PmcA, but completely abolished the GvpE-mediated activation (Fig. 4b
). In summary, these results demonstrated that the sequence upstream of BRE had no influence on the basal promoter activity, but the GvpE-mediated activation was negatively affected when the sequence TGAACCAA-n4-TGAAACGG was altered.
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DISCUSSION |
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We investigated whether or not the PcA-TATA box and PcA-BRE element were the reason for the undetectable basal activity of PcA by substituting these elements with the respective sequences of PpA which exhibit the strongest basal promoter activity of the three PA promoters. Even the substitution of both PcA promoter elements by the respective PpA promoter sequences resulted in a promoter with no detectable basal promoter activity, demonstrating that both PpA elements were not sufficient to drive the basal transcription of these PcA-pA promoters. Since the exchange of an additional 21 bp of PcA sequences further upstream with the respective PpA sequences did not result in a measurable basal transcription, the sequence located between the TATA box and the transcription start must be responsible for the observed lack of basal transcription in PcA. Earlier results have shown that alterations in the respective sequences of the PpA promoter strongly affect the basal transcription (Hofacker et al., 2004). From these results we assume that the higher GC content found in PcA compared to PmcA and PpA (13 of 22 bp versus 9 of 22 bp, see Fig. 1
) interferes with the open complex formation of the RNA polymerase and might be the major reason for the undetectable basal transcription in PcA.
In the presence of GvpE the promoter derivatives +10u, +5u, 5u, 10u led to enhanced BgaH activities, similar to the PpA-cA-bgaH construct described earlier (Gregor & Pfeifer, 2001). These four promoter variants were also activated by the heterologous pGvpE and mcGvpE proteins (to a minor extent), whereas the original PcA-bgaH construct is only induced by the homologous cGvpE (Gregor & Pfeifer, 2001
). In contrast, mutants 5d and 10d harbouring the original PcA-BRE were only induced by cGvpE. These results led to the conclusion that the PpA-BRE element was a major reason for the enhanced and extended promoter activities observed with the four PpA-cA mutants mentioned above. The PpA-BRE could cause a stronger binding of the original TFB protein (or even bind a different TFB protein), resulting in the enhanced GvpE-mediated activation.
The PmcA promoter of Hfx. mediterranei was selected to determine the sequences required for GvpE-mediated activation. PmcA yields a basal promoter activity and also has the strongest GvpE-induction of all PA promoters. A 4 bp scanning mutagenesis was done throughout the 34 bp region separating the two BRE elements of the oppositely oriented PmcA and PmcD promoters. The region between the TATA box and the transcription start site was not included, since alterations in the related region of the PpA promoter did not affect the GvpE-mediated activation (Hofacker et al., 2004). None of these mutations in PmcDPmcA affected the basal PmcA promoter activity. With respect to the GvpE-mediated activation, mutants carrying alterations of the sequences TGAACCAA adjacent to BRE and TGAAACGG located further upstream showed a reduced GvpE-induced promoter activity, demonstrating that both these sequences were important for the GvpE-mediated activation. These two sequences were similar. An alignment of the three PDPA regions of p-vac, c-vac and mc-vac indicated three different conserved regions of >4 bp, two of which are part of this sequence element (Fig. 5a
, conserved nucleotides are marked in bold). An alignment of all 8 nt sequence elements led to the consensus sequence TGAAACNA (Fig. 5b
). A similar sequence element can be determined with respect to PmcD (which is also activated by GvpE). This sequence includes the conserved sequence ATTAC close to the BRE element of PmcD and PpD (see Fig. 5a
). These two promoters are activated by GvpE, whereas the PcD promoter of the c-vac region is not responsive to GvpE-mediated activation, presumably because the PpD-BRE element is located 10 bp further away (Krüger & Pfeifer, 1996
). However, this needs further proof. A footprint analysis would be very helpful, but the GvpE binding sites determined by these in vivo analyses cover almost the entire PmcDPmcA region. The palindrome sequence GTTG-n6-ACCA, originally hypothesized as the GvpE-binding site by Krüger & Pfeifer (1996)
, did not contribute to the GvpE-mediated activation. Although mutations in the ACCA portion of this sequence in the course of the 4 bp scanning mutagenesis in PmcA resulted in a reduced GvpE-mediated promoter activation, mutations in the GTTG portion of this palindrome had no influence on the promoter activation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 August 2004;
revised 30 September 2004;
accepted 8 October 2004.
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