Institut für Mikrobiologie und Genetik, Technische Universität Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany1
Author for correspondence: Felicitas Pfeifer. Tel: +49 6151 162957. Fax: +49 6151 162956. e-mail: pfeifer{at}bio.tu-darmstadt.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: Haloferax volcanii, ß-galactosidase reporter gene, gene regulation
Abbreviations: gvp, gas vesicle protein gene; Gvp, gas vesicle protein
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We are using the genes encoding gas vesicles to study gene regulation in halophilic archaea. Gas vesicle formation involves the 14 genes gvpDEFGHIJKLM and gvpACNO that cluster in a genomic region called the vac region. Three different vac regions have been characterized, two of these are found in Halobacterium salinarum PHH1, namely the chromosomal c-vac region and the plasmid-borne p-vac region of the 150 kb plasmid pHH1 (Englert et al., 1992a ). In the case of the related strain Hb. salinarum NRC-1, two identical gvp1 gene clusters corresponding to the p-vac region are present on the two large plasmids pNRC100 and pNRC200, and an additional gvp2 gene cluster almost identical to c-vac is found on pNRC200 (DasSarma et al., 1994
; Ng et al., 1998
, 2000
). The mc-vac region (mediterranei chromosomal) is found in the chromosome of Haloferax mediterranei (Englert et al., 1992a
). We use the designations c-gvp, p-gvp and mc-gvp to distinguish the various gvp genes according to the origin of the vac region. The c-vac region is expressed only in p-vac deletion strains such as Hb. salinarum PHH4 harbouring the 35 kb plasmid pHH4 (Pfeifer & Blaseio, 1989
).
The gvpACNO gene cluster encodes the major structural protein, GvpA, of the gas vesicles, and GvpC, a protein involved in stabilization of the structure and in shape formation (Offner et al., 1996 ). The products of the gvpD and gvpE genes are involved in regulation: GvpE is a transcriptional activator (Krüger & Pfeifer, 1996
; Krüger et al., 1998
), whereas GvpD takes part in the repression of gas vesicle formation (Englert et al., 1992b
; Pfeifer et al., 1994
, 2001
). These two Gvp proteins predominantly influence the promoter of the gvpACNO unit and thus determine whether and when gas vesicles are formed. The promoter of the p-gvpA gene of Hb. salinarum PHH1 has a high activity throughout growth, and transcription results in large amounts of p-gvpA mRNA plus minor amounts of p-gvpACNO cotranscripts (Horne et al., 1991
; Englert et al., 1992a
). In contrast, the promoter of the c-gvpA gene is active only during the stationary phase in Hb. salinarum PHH4 (Horne & Pfeifer, 1989
), similar to the promoter of the mc-gvpA gene of Hf. mediterranei (Englert et al., 1992a
).
The activities of the c-gvpA and mc-gvpA promoters depend on the presence of the GvpE activator protein, as demonstrated by transformation experiments using Haloferax volcanii as recipient. This strain does not contain the gvp genes and offers a clean genetic background. Transformants harbouring the p-gvpA gene produce p-gvpA mRNA throughout growth, whereas the respective c-gvpA and mc-gvpA transformants contain no or minor amounts of gvpA mRNA. However, transformants harbouring c-gvpA plus c-gvpE (expressed under fdx promoter control) contain large amounts of c-gvpA mRNA and also GvpA protein (Krüger et al., 1998 ). Similarly, mc-gvpADE transformants produce large amounts of mc-gvpA transcripts and GvpA protein (Röder & Pfeifer, 1996
). These results demonstrate that GvpE is a transcriptional activator protein. Homology modelling of the C-terminal part of GvpE indicates an amphiphilic
helix that exhibits a similar structure as the one found in the basic leucine zipper (bZIP) activator protein GCN4 of yeast (Ellenberger et al., 1992
; Krüger et al., 1998
). A region adjacent to the N terminus consists of basic amino acids and constitutes a putative DNA-binding site. Such bZIP proteins are transcriptional activators predominantly found in eukarya.
To investigate the basal and induced activities of the three different gvpA promoters in Hf. volcanii transformants in more detail, we tested a halobacterial ß-galactosidase as reporter. The bgaH gene encoding this enzyme has been isolated from a superblue mutant of Haloferax alicantei (Holmes et al., 1997 ; Holmes & Dyall-Smith, 2000
). Hf. volcanii lacks detectable ß-galactosidase activity and is thus suitable for these studies. Transformants expressing the enzyme turn blue when sprayed with X-Gal on agar plates, and the ß-galactosidase activity can be quantified by a standard assay hydrolysing ONPG. An application of this system for the investigation of various promoters in Hb. salinarum has recently been reported by Patenge et al. (2000)
.
In this report, Hf. volcanii transformants were produced containing fusions between the various gvpA promoter regions and the bgaH reading frame. Different fusion constructs were tested at the level of mRNA formation and at the level of ß-galactosidase activity to gain insights into the amount of both products. ß-Galactosidase activity did not always reflect the mRNA data, indicating that the fusion site is rather crucial for the translation of the bgaH reading frame. However, the AbgaH fusion genes were sufficient to investigate gvpA regulation.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Constructs used for transformation.
The 2203 bp bgaH reading frame was amplified by PCR using the oligonucleotides bgaH-NcoI and bgaH-BamHI (Table 1), together with pMLH32 as template (Holmes & Dyall-Smith, 2000
). The NcoI site of primer bgaH-NcoI included the ATG start codon of the bgaH reading frame, whereas the bgaH-BamHI primer was complementary to a sequence located 200 bp downstream of the bgaH stop codon. Each gvpA promoter region was amplified by PCR as a XbaINcoI fragment using synthetic oligonucleotides (Table 1
) and subfragments of the three different vac regions inserted in E. coli plasmids as templates. For the fusion at the ATG start codon of gvpA, the respective A-XbaI primer was used, together with pA1-NcoI (amplification of the 109 bp pA1 promoter fragment), cA1-NcoI (109 bp cA1 promoter fragment), and mcA1-NcoI (119 bp mcA1 promoter fragment) (see Fig. 1
). Slightly larger fragments spanning the ATG start codon of gvpA were amplified using the respective A-XbaI primer together with pA-NcoI (resulting in the 127 bp pA promoter fragment), cA-NcoI (137 bp cA promoter fragment) and mcA-NcoI (126 bp mcA promoter fragment). For the chimeric pAcA promoter fragment, a 60 nt megaprimer was amplified using the primers cA-XbaI and pAcA and the c-gvpA gene as template. The resulting megaprimer included 21 bp of the p-gvpA promoter sequence fused to the TATA box of the c-gvpA gene; this megaprimer was used together with primer cA-NcoI in the second PCR to amplify the entire pAcA promoter fragment.
|
|
Transformation of Hf. volcanii WFD11.
Prior to the transformation of Hf. volcanii, each construct was passaged through E. coli GM1674 (dam-negative) to avoid a halobacterial restriction barrier (Holmes et al., 1991 ). Transformation was carried out as described previously (Pfeifer & Ghahraman, 1993
). The presence and the amount of the desired plasmid(s) in each transformant was determined by Southern analyses using specific DNA probes. A 3·5 kb HindIII/BamHI fragment derived from pMLH32 was used for bgaH, whereas 600700 bp Acc65I/NcoI fragments containing the respective gvpE reading frame were used to generate the gvpE-specific probes. Each probe was labelled with digoxigenin using the DIG-Labelling Kit from Roche.
RNA isolation and transcript analysis.
Total RNA was isolated by the method of Chomczynski & Sacchi (1987) , or by using the RNeasy Kit from Qiagen, followed by DNase I digestion. For transcript analysis, 5 or 10 µg RNA was separated on denaturing, formaldehyde-containing 1·2% (w/v) agarose gels, followed by transfer to nylon membranes (Ausubel et al., 1988
). A strand-specific bgaH RNA probe was synthesized with the T3/T7 system using the 2·2 kb NcoIBamHI bgaH fragment inserted in pBluescript as template. The probe was labelled with digoxigenin using the DIG RNA Labelling Kit obtained from Roche.
ß-Galactosidase assay.
ß-Galactosidase activity in colonies was visualized by spraying the transformant colonies grown on agar plates with X-Gal (10 mg ml-1). ß-Galactosidase activity in cell lysates was measured using the ONPG assay as described by Holmes et al. (1997) . The protein concentration was determined by the Bradford assay (Ausubel et al., 1988
) using BSA as standard.
Accession numbers of DNA sequences.
These are as follows: U70664 (bgaH gene), X94688 (c-vac region of Hb. salinarum PHH4), X64701 (mc-vac region of Hf. mediterranei), X64729 (p-gvpACNO) and X55648 (p-gvpDEFGHIJKLM). The latter two sequences derive from the p-vac region on plasmid pHH1 of Hb. salinarum PHH1 (formerly Hb. halobium NRC817).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate the regulatory elements that contribute to the expression of the gvpA genes in Hf. volcanii transformants in more detail, we amplified fragments containing the respective gvpA promoter regions, including the region transcribed into the mRNA leader (Fig. 1b). The first round of amplifications resulted in the fragments pA1, cA1 and mcA1 (109119 bp) containing the ATG start codon of the respective gvpA gene within the NcoI cloning site. The second series of amplifications resulted in the 15-nt-larger pA, cA and mcA fragments (see Fig. 1b
). The 2203 bp bgaH reading frame, amplified as a NcoI/BamHI fragment, was fused to the respective A1- and A-promoter fragments at the ATG start codon of bgaH. The A1bgaH fusions encoded the native ß-galactosidase, whereas the AbgaH fusions encoded a ß-galactosidase protein with five additional amino acids derived from GvpA at the N terminus (MAQPD or MVQPD, see below). Constructs containing the A1bgaH or AbgaH fusion genes were used to transform Hf. volcanii. Transformants containing only the AbgaH (or A1bgaH) construct were used to monitor the basal amount of mRNA and ß-galactosidase activity, whereas transformants harbouring in addition one of the three different gvpE-pJAS constructs (i.e. AbgaH/E or A1bgaH/E transformants) were used to determine the GvpE-dependent activity of each gvpA regulatory region.
Reporter gene fusion at the ATG start codon of the gvpA reading frame
The various A1bgaH and A1bgaH/E transformants were analysed by Southern blotting for the presence of the desired construct(s). The desired transformants were streaked as cross-shaped colonies on agar plates and sprayed with X-Gal solution after growth to demonstrate ß-galactosidase activity (Fig. 2). Colonies of Hf. volcanii are usually orange-red and retain this colour after X-Gal treatment, similar to Hf. volcanii containing the c-gvpE gene (cE, Fig. 2
). The transformants containing the original bgaH construct pMLH32 (Holmes & Dyall-Smith, 2000
) turned dark blue (bgaH, Fig. 2
). Transformants containing the mcA1bgaH construct (mcA1) formed brownish red colonies, consistent with the very low activity of the mc-gvpA promoter in transformants harbouring the mc-gvpA gene (Röder & Pfeifer, 1996
). The mcA1bgaH/cE transformants (mcA1bgaH plus c-gvpE-pJAS) turned dark blue, demonstrating the induction of mcA1bgaH expression by the cGvpE protein (mcA1/cE, Fig. 2
). The cA1bgaH transformants (cA1) remained orange-red, which is indicative of the inactive c-gvpA promoter, similiar to the complete lack of detectable amounts of c-gvpA mRNA in transformants containing the c-gvpA gene (Krüger & Pfeifer, 1996
). However, colonies of the cA1bgaH/cE transformants (cA1/cE) also remained orange-red, demonstrating that these cells did not contain detectable amounts of ß-galactosidase activity (Fig. 2
). This result was in contrast to the data obtained with c-gvpA+c-gvpE-pJAS transformants which produce large amounts of c-gvpA mRNA and GvpA protein (Krüger et al., 1998
). Also, transformants containing the pA1bgaH construct (pA1) remained red although the p-gvpA gene is transcribed in p-gvpA transformants. Since the cA1bgaH and pA1bgaH genes contained the entire promoter and regulatory region, including the transcriptional start site of the respective gvpA gene, these results raised the question of whether the lack of ß-galactosidase activity was caused by transcriptional or translational problems.
|
|
The expression of the different AbgaH genes in transformants was investigated with each of the three GvpE proteins, and the specific ß-galactosidase activities were quantified in samples taken throughout the growth period using the ONPG assay. The analyses of the transformants harbouring the mcAbgaH construct by itself or including one of the three gvpE genes (mcA, mcA/pE, mcA/cE, and mcA/mcE transformants) is presented in Fig. 4; similar experiments were done with all other transformants (data not shown). Since the ß-galactosidase activities increased during exponential and early stationary phases, a mean activity was calculated from the activities determined in samples taken between 100 and 150 h of growth (Table 2
). The highest specific ß-galactosidase activity determined among the non-induced AbgaH genes was found with pAbgaH transformants; mcAbgaH transformants showed a low activity, whereas cAbgaH transformants did not contain detectable ß-galactosidase activity (Table 2
). The activation of each AbgaH gene by GvpE was tested in the three different AbgaH/E transformants. The various mcAbgaH/E transformants demonstrated high ß-galactosidase activities (ranging from 120 to 900 mU mg-1), with pGvpE as the weakest and cGvpE as the strongest activator protein (Table 2
). The pAbgaH gene was also activated by all three GvpE proteins, but the expression of cAbgaH was only achieved with the homologous cGvpE protein (cAbgaH/cE transformants, Table 2
). The specific ß-galactosidase activity determined in the latter transformant was slightly higher than the basal ß-galactosidase activity in the pAbgaH transformants. None of the heterologous GvpE activator proteins was able to activate the cAbgaH gene (Table 2
). In each case, the cGvpE protein appeared to be the strongest transcriptional activator protein among the three GvpE proteins (Table 2
).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reporter gene fusions at the ATG start codon of the gvpA gene may lead to problems in translation
All A1bgaH and A1bgaH/cE transformants contained the expected amount of bgaH mRNA. The mcA1bgaH, cA1bgaH and mcA1bgaH/cE transformants also showed ß-galactosidase activity, indicating that the mRNA consisting of the gvpA mRNA leader and the bgaH reading frame was indeed translated. However, the pA1bgaH and cA1bgaH/cE transformants did not show detectable amounts of ß-galactosidase activity despite the presence of the mRNA, suggesting that a problem occurred at the translational level. A single point mutation (T or AC) was introduced 5' to the ATG start codon during the construction of the NcoI site for the fusion (Fig. 1a
). Presumably, this alteration must be the reason for the lack of translation of these pA1 and cA1bgaH mRNAs. However, a similar alteration was introduced in the mcA1bgaH mRNA without preventing ß-galactosidase formation. The leader sequences of the gvpA mRNAs are similar but not identical, and it is still unclear which regions are important for the initiation of translation. It is possible that the mutations destroyed a signal important for translation initiation, or forced an mRNA secondary structure that masked the AUG start codon. Various stemloop secondary structures are possible in the leader region that are currently under investigation. Regardless, these results imply that the fusion of the bgaH reporter gene should not be made at the start codon of the gene under investigation.
The ß-galactosidase activity reflects the activity of the gvpA promoters
The larger promoter fragments used for the pA, cA and mcAbgaH constructs revealed the expected mRNAs that, when present, were translated into ß-galactosidase. The analysis of the product formation in each AbgaH transformant indicated no mRNA and ß-galactosidase in the cAbgaH transformant, a low mRNA amount and ß-galactosidase activity in the mcAbgaH, and a high mRNA amount and ß-galactosidase activity in the pAbgaH transformant. These results reflected the activities of the various gvpA promoters in gvpA transformants (Röder & Pfeifer, 1996 ; Krüger et al., 1998
). Each promoter of the AbgaH genes was stimulated at least by the homologous GvpE protein, indicating that the binding site of GvpE is contained within the promoter fragments used for the construction. The promoter of the mc-gvpA gene was the strongest activated promoter tested. Although the basal ß-galactosidase activity was low in mcAbgaH transformants, the activity was stimulated 80-fold by the homologous mcGvpE protein and 150-fold by the cGvpE protein. Among the various GvpE proteins tested with the mc-gvpA promoter, pGvpE appeared to be the weakest and cGvpE the strongest activator. The pAbgaH construct was also stimulated by all three GvpE proteins in respective pAbgaH/E transformants, but the overall induction was lower, except for the homologous pGvpE protein. Also, in this case, cGvpE appeared to be the strongest activator. It is possible that the single amino acid difference in the GvpA sequence near the N terminus results in a difference in the specific activity of the two ß-galactosidase proteins (MVQPD in the case of mcAbgaH and MAQPD in the case of cA and pAbgaH). However, the products of the pAbgaH and the cAbgaH genes are identical, and at least these specific ß-galactosidase activities can be compared with each other.
Similar to the c-gvpA gene, the cAbgaH fusion gene was completely inactive in transformants. Only the homologous cGvpE activator was able to induce mRNA and ß-galactosidase formation, but the activity remained relatively low. The pGvpE and mcGvpE proteins were unable to induce the c-gvpA promoter, which could be due to a weaker activity, but the DNA sequence responsible for GvpE binding could also contribute. The inability of pGvpE to activate the c-gvpA promoter has already been recognized during heterologous complementation studies using the p-gvpDEFGHIJKLM unit of the p-vac region to complement the c-gvpACNO genes in Hf. volcanii transformants: no GvpA formation could be observed, suggesting that pGvpE cannot activate the c-gvpA promoter (Offner et al., 1998 ). This could also be the reason that gas vesicles of Hb. salinarum PHH1 are only formed by pGvpA: the pGvpE activator protein produced from the p-vac region is unable to stimulate the expression of the c-gvpACNO gene cluster present in the same cell. The latter gvp gene cluster is only activated by the homologous cGvpE activator protein.
Archaeal promoters consist of a TATA box centred around position -28 relative to the transcriptional start site, and many promoters contain the TFB recognition element BRE (consensus: RNWAAW, R=A or G, N=any base, W=A or T) (Bell et al., 1999 ). The p-gvpA promoter contains a TATA box almost identical to the archaeal consensus, and also a highly conserved putative BRE element (Fig. 1a
). These features could be the reason for the relatively high basal activity of the p-gvpA promoter, which does not depend on an activator protein for expression. In contrast, the inactive c-gvpA promoter shows less conservation in the TATA box, and no sequences reminiscent of a BRE element (Fig. 1a
). Activation of this promoter required the cGvpE activator protein; neither pGvpE nor mcGvpE were sufficient for activation.
For a preliminary investigation of the DNA sequences required for GvpE stimulation, a chimeric pAcA promoter was tested for bgaH expression in transformants. This promoter fragment consisted mainly of c-gvpA sequences, but 21 nt 5' to the TATA box were substituted by the respective sequences derived from the p-gvpA promoter, including the almost perfect BRE element (Fig. 1a). The pAcAbgaH transformants indicated no basal ß-galactosidase activity, suggesting that the putative BRE element next to the TATA box was not sufficient for transcription initiation at this promoter. However, the pAcAbgaH construct was induced by all three GvpE proteins in the respective transformants, and cGvpE was the strongest activator. It is still not known where the GvpE activator protein(s) bind in the promoter region for activation, but the fact that all GvpE proteins were able to activate the pAcAbgaH gene suggested that the sequences upstream of the TATA box were important in this process. Mutagenesis of the entire promoter region is in progress to determine overall promoter strength and the nucleotides important for the activation by GvpE.
The ß-galactosidase activity is more stable than the bgaH mRNA
The investigation of the amount of bgaH mRNA in the mcAbgaH/cE transformant indicated a high amount of mRNA during the exponential and early stationary phases, and no bgaH mRNA in the late stationary phase. The increase in the ß-galactosidase activity was in accordance with the increase in the mRNA during the exponential phase, but stayed at a high level during the stationary phase, implying that the ß-galactosidase protein is rather stable. Thus, the bgaH reporter system is useful to determine the initial promoter activation. However, since the bgaH mRNA and ß-galactosidase stabilities deviate from each other, promoter de-activation studies are not possible. This observation contrasts with results presented by Patenge et al. (2000) , where the promoter of the bacterio-opsin (bop) gene and various fdx promoter mutants were analysed with the bgaH reporter system in the related species Hb. salinarum. In the case of the bop promoterbgaH fusion, the ß-galactosidase activity remained at a high level for 96 h, but was reduced in the single sample taken after 120 h. However, other transformants contained stable amounts of ß-galactosidase activity (Patenge et al., 2000
).
Overall, the bgaH reporter gene has turned out to be a useful tool for the investigation of promoter and regulatory activities at a more quantitative level. Although Hf. volcanii is only distantly related, halobacterial promoters derived from Hf. mediterranei and Hb. salinarum can be studied and compared to each other in this system. In further experiments, we hope to gain more insights into the activation of the various gvpA promoters by GvpE, and study the action of other regulatory proteins encoded by the vac region.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baglia, N., Goo, Y. A., Ng, W. V., Hood, L., Daniels, C. & DaSarma, S. (2000). Is gene expression in Halobacterium NRC-1 regulated by multiple TBP and TFB transcription factors? Mol Microbiol 36, 1184-1185.[Medline]
Bell, S., Jaxel, C., Nadal, M., Kosa, P. & Jackson, S. (1998). Temperature, template topology, and factor requirements of archaeal transcription. Proc Natl Acad Sci USA 95, 15218-15222.
Bell, S., Kosa, P., Sigler, P. & Jackson, S. (1999). Orientation of the transcription preinitiation complex in Archaea. Proc Natl Acad Sci USA 96, 13662-13667.
Chomczynski, P. & Sacchi, N. (1987). Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156-159.[Medline]
Cline, S. W., Schalkwyk, L. C. & Doolittle, W. F. (1989). Transformation of the archaebacterium Halobacterium volcanii with genomic DNA. J Bacteriol 171, 4987-4991.[Medline]
DasSarma, S., Arora, P., Lin, F., Molinari, E. & Yin, L. (1994). Wild-type gas vesicle formation requires at least ten genes in the gvp gene cluster of Halobacterium halobium plasmid pNRC100. J Bacteriol 176, 7646-7652.[Abstract]
Ellenberger, T. E., Brandl, C. J., Struhl, K. & Harrison, S. C. (1992). The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted helices: crystal structure of the proteinDNA complex. Cell 71, 1223-1237.[Medline]
Englert, C., Horne, M. & Pfeifer, F. (1990). Expression of the major gas vesicle protein in the halophilic archaebacterium Haloferax mediterranei is modulated by salt. Mol Gen Genet 222, 225-232.[Medline]
Englert, C., Krüger, K., Offner, S. & Pfeifer, F. (1992a). Three different but related gene clusters encoding gas vesicles in halophilic archaea. J Mol Biol 227, 586-592.[Medline]
Englert, C., Wanner, G. & Pfeifer, F. (1992b). Functional analysis of the gas-vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac-region boundary and suggests a regulatory role for the gvpD gene or its product. Mol Microbiol 6, 3543-3550.[Medline]
Hausner, W., Wettach, J., Hethke, C. & Thomm, M. (1996). Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase. J Biol Chem 271, 30144-30148.
Holmes, M. L. & Dyall-Smith, M. (2000). Sequence and expression of a halobacterial ß-galactosidase gene. Mol Microbiol 36, 114-122.[Medline]
Holmes, M. L., Nuttall, S. D. & Dyall-Smith, M. (1991). Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase. J Bacteriol 12, 3807-3813.
Holmes, M. L., Scopes, R., Moritz, R., Simpson, R., Englert, C., Pfeifer, F. & Dyall-Smith, M. (1997). Purification and analysis of an extremely halophilic ß-galactosidase from Haloferax alicantei. Biochim Biophys Acta 1337, 276-286.[Medline]
Horne, M. & Pfeifer, F. (1989). Expression of two gas vacuole protein genes in Halobacterium halobium and other related species. Mol Gen Genet 218, 437-444.[Medline]
Horne, M., Englert, C., Wimmer, C. & Pfeifer, F. (1991). A DNA region of 9 kb contains all genes necessary for gas vesicle synthesis in halophilic archaebacteria. Mol Microbiol 5, 1159-1174.[Medline]
Krüger, K. & Pfeifer, F. (1996). Transcript analysis of the c-vac region, and differential synthesis of the two regulatory gas-vesicle proteins GvpD and GvpE in Halobacterium salinarium PHH4. J Bacteriol 178, 4012-4019.[Abstract]
Krüger, K., Hermann, T., Armbruster, V. & Pfeifer, F. (1998). The transcriptional activator GvpE for the halobacterial gas vesicle genes resembles a basic region leucine-zipper regulatory protein. J Mol Biol 279, 761-771.[Medline]
Lam, W. L. & Doolittle, W. F. (1989). Shuttle vectors for the archaebacterium Halobacterium volcanii. Proc Natl Acad Sci USA 86, 5478-5482.[Abstract]
Ng, W. L., Ciufo, S., Smith, T. & 9 other authors (1998). Snapshot of a large dynamic replicon in a halophilic archaeon: megaplasmid or minichromosome? Genome Res 8, 11311141.
Ng, W. L., Kenney, S., Mahairas, G. & 14 other authors (2000). Genome sequence of Halobacterium species NRC-1. Proc Natl Acad Sci USA 97, 1217612181.
Offner, S., Wanner, G. & Pfeifer, F. (1996). Functional studies of the gvpACNO operon of Halobacterium salinarium reveal that the GvpC protein shapes gas vesicles. J Bacteriol 178, 2071-2078.[Abstract]
Offner, S., Ziese, U., Wanner, G., Typke, D. & Pfeifer, F. (1998). Structural characteristics of halobacterial gas vesicles. Microbiology 144, 1331-1342.[Abstract]
Palmer, B. & Marinus, M. (1994). The dam and dcm strains of Escherichia coli a review. Gene 143, 1-12.[Medline]
Patenge, N., Haase, A., Bolhuis, H. & Oesterhelt, D. (2000). The gene for a halophilic ß-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum. Mol Microbiol 36, 102-113.
Pfeifer, F. & Blaseio, U. (1989). Insertion elements and deletion formation in a halophilic archaebacterium. J Bacteriol 171, 5135-5140.[Medline]
Pfeifer, F. & Ghahraman, P. (1993). Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region, the gas-vesicle gene cluster and insertion elements. Mol Gen Genet 238, 193-200.[Medline]
Pfeifer, F., Offner, S., Krüger, K., Ghahraman, P. & Englert, C. (1994). Transformation of halophilic archaea and investigation of gas-vesicle synthesis. Syst Appl Microbiol 16, 569-577.
Pfeifer, F., Zotzel, J., Kurenbach, B., Röder, R. & Zimmermann, P. (2001). A p-loop motif and two basic regions in the regulatory protein GvpD are important for the repression of gas vesicle formation in the archaeon Haloferax mediterranei. Microbiology 147, 63-73.
Qureshi, S. & Jackson, S. (1998). Sequence-specific DNA binding by the S. shibatae TFIIB homolog, TFB, and its effect on promoter strength. Mol Cell 1, 389-400.[Medline]
Qureshi, S., Baumann, P., Rowlands, T., Khoo, B. & Jackson, S. (1995). Cloning and functional analysis of the TATA binding protein from Sulfolobus shibatae. Nucleic Acids Res 23, 1775-1781.[Abstract]
Reeve, J. N., Sandman, K. & Daniels, C. (1997). Archaeal histones, nucleosomes, and transcription initiation. Cell 89, 999-1002.[Medline]
Röder, R. & Pfeifer, F. (1996). Influence of salt on the transcription of the gas-vesicle genes of Haloferax mediterranei and identification of the endogenous transcriptional activator gene. Microbiology 142, 1715-1723.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Thomm, M. (1996). Archaeal transcription factors and their role in transcription initiation. FEMS Microbiol Rev 18, 159-171.[Medline]
Thompson, D. K., Palmer, J. R. & Daniels, C. J. (1999). Expression and heat-responsive regulation of a TFIIB homologue from the archaeon Haloferax volcanii. Mol Microbiol 33, 1081-1092.[Medline]
Zillig, W., Palm, P., Klenk, H.-P., Langer, D., Hüdepohl, U., Hain, J., Lanzendörfer, M. & Holz, I. (1993). Transcription in archaea. In The Biochemistry of Archaea , pp. 367-391. Edited by M. Kates. Amsterdam:Elsevier Science.
Received 18 January 2001;
revised 15 March 2001;
accepted 19 March 2001.