Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea

Dagmar Gregor1 and Felicitas Pfeifer1

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The bgaH reading frame encoding a ß-galactosidase of ‘Haloferax alicantei’ was used as a reporter gene to investigate three different promoter regions derived from gvpA genes of Haloferax mediterranei (mc-gvpA) and Halobacterium salinarum (c-gvpA and p-gvpA) in Haloferax volcanii transformants. The fusion of bgaH at the start codon of each gvpA reading frame (A1–bgaH fusion genes) caused translational problems in some cases. Transformants containing constructs with fusions further downstream in the gvpA reading frame (A–bgaH) produced ß-galactosidase, and colonies on agar plates turned blue when sprayed with X-Gal. The ß-galactosidase activities quantified by standard ONPG assays correlated well with the mRNA data determined with transformants containing the respective gvpA genes: the cA–bgaH fusion gene was completely inactive, the mcA–bgaH transformants showed low amounts of products, whereas the pA–bgaH fusion gene was constitutively expressed in the respective transformants. The transcription of each A–bgaH gene was activated by the homologous transcriptional activator protein GvpE. The cGvpE, pGvpE and mcGvpE proteins were able to activate the promoter of pA–bgaH and mcA–bgaH, whereas the promoter of cA–bgaH was only activated by cGvpE. Among the three GvpE proteins tested, cGvpE appeared to be the strongest transcriptional activator.

Keywords: Haloferax volcanii, ß-galactosidase reporter gene, gene regulation

Abbreviations: gvp, gas vesicle protein gene; Gvp, gas vesicle protein


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Halophilic archaea are model organisms to study archaeal gene regulation in vivo. They are easy to grow, and a transformation system including vector plasmids conferring resistance to mevinolin or novobiocin is available (Lam & Doolittle, 1989 ; Holmes et al., 1991 ). Transcription in archaea possesses fundamental similarities to eukaryal transcription: archaeal promoters contain a TATA box element centred 25–28 nucleotides upstream of the transcription start site, and gene expression involves a single DNA-dependent RNA polymerase comprising 12 subunits that are homologous to the core components of the eukaryal RNA polymerase II (Zillig et al., 1993 ). Two additional factors, namely the TATA-box-binding protein TBP and TFB (the archaeal homologue of transcription factor TFIIB) are sufficient for the initiation of basal transcription (Hausner et al., 1996 ; Qureshi et al., 1995 ; Thomm, 1996 ; Reeve et al., 1997 ). TFB recognizes a DNA sequence immediately upstream of the TATA box (the TFB recognition element BRE found in many archaeal genes) as demonstrated by in vitro transcription studies using recombinant factors and RNA polymerase from the thermophilic archaeon Sulfolobus (Qureshi & Jackson, 1998 ; Bell et al., 1998 , 1999 ). So far, in vitro transcription systems have been established for some methanogenic and thermophilic archaea, but not for halophilic archaea. The genome sequences of Methanococcus jannaschii and Methanobacterium thermautotrophicum contain single genes for TBP and TFB proteins, whereas halophilic archaea harbour multiple gene copies for both TBP and TFB (Ng et al., 1998 ; Baglia et al., 2000 ; Thompson et al., 1999 ).

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 {alpha} 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 A–bgaH fusion genes were sufficient to investigate gvpA regulation.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Growth conditions.
Escherichia coli strains DH5{alpha}, XL-1 Blue (Stratagene) and GM1674, a dam-negative strain (Palmer & Marinus, 1994 ) were grown aerobically at 37 °C in LB broth (Sambrook et al., 1989 ). For selection of transformants, ampicillin was added at a concentration of 100 µg ml-1. Hf. volcanii WFD11, lacking the endogenous plasmid pHV2 (Cline et al., 1989 ), was grown in rich medium containing (l-1): 175 g NaCl, 37 g MgSO4 . 7 H2O, 3·7 g KCl, 5 g Bacto tryptone, 3 g Bacto yeast extract, 25 ml 1 M Tris/HCl pH 7·2, 5 ml 10% CaCl2 . H2O and 100 µl 100 µM MnCl2. Transformants were selected on agar plates containing 0·2 µg novobiocin ml-1 and/or 6 µg mevinolin ml-1. Lovastatin (a derivative of mevinolin) was a gift from MSD Sharpe & Dohme.

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 XbaI–NcoI 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.


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Table 1. Synthetic oligonucleotides used for the amplification by PCR

 


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Fig. 1. (a) Promoter region of the three gvpA genes and of the pAcA hybrid, and (b) extent of the A1- and A-promoter fragments. (a) The start site of transcription (G) is labelled +1, and the ATG start codon is shown in bold. The TATA box centred around -28 is shown in bold. The consensus sequences of the BRE element and of the TATA box are given above (R=A or G, N=any base, W=A or T, Y=C or T). Sequences derived from the p-gvpA promoter in the pAcA–bgaH promoter are underlined. The nucleotide sequences around the ATG start codon used to introduce the NcoI site (CCATGG) during the construction of the A1–bgaH gene fusions are shown in italic. (b) Schematic representation of the gvpA promoter region. including the TATA box and the putative BRE element as well as the start sites of transcription (+1) and of translation (ATG). The bars below the map indicate the various oligonucleotides used for the PCR amplifications. The lines above the map represent the promoter fragments pA1, cA1 and mcA1, or the slightly larger fragments pA, cA and mcA.

 
Each promoter fragment was purified by agarose gel electrophoresis, cleaved with XbaI/NcoI, and ligated with the bgaH reading frame (cleaved with NcoI and BamHI) and the XbaI/BamHI-hydrolysed E. coli vector pBluescript II SK(+) (Stratagene). The exact fusion in each A1–bgaH and A–bgaH gene was confirmed by DNA sequence analysis. The A1–bgaH and A–bgaH genes were transferred as XbaI/BamHI fragments into the halobacterial vector plasmid pWL102 (Lam & Doolittle, 1989 ). For the expression of the p-gvpE and mc-gvpE reading frames, both were amplified by PCR using the primer pairs pE1/pE2, or mcE1/mcE2 (Table 1). The PCR products were inserted into the halobacterial expression vector pJAS35 for expression under fdx promoter control (Pfeifer et al., 1994 ). The c-gvpE-pJAS construct has already been described (Krüger et al., 1998 ).

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 600–700 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 NcoI–BamHI 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Three different gvpA genes encoding the major gas vesicle structural protein of Hb. salinarum and Hf. mediterranei were used for a comparative investigation of gene expression. These genes are transcribed as 340 nt mRNAs containing a 19–20 nt leader region (Horne & Pfeifer, 1989 ; Englert et al., 1990 ). The gvpA mRNA leader regions are almost identical except for a 4 nt sequence at position 5–8 of the mRNA, and the nucleotides adjacent to the ATG start codon (Fig. 1a). No sequences reminiscent of a Shine–Dalgarno motif are present. The mRNA of the bgaH gene of ‘Hf. alicantei also contains a 34 nt leader region with no obvious Shine–Dalgarno motif (Holmes & Dyall-Smith, 2000 ).

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 (109–119 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 A1–bgaH fusions encoded the native ß-galactosidase, whereas the A–bgaH 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 A1–bgaH or A–bgaH fusion genes were used to transform Hf. volcanii. Transformants containing only the A–bgaH (or A1–bgaH) 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. A–bgaH/E or A1–bgaH/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 A1–bgaH and A1–bgaH/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 mcA1–bgaH 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 mcA1–bgaH/cE transformants (mcA1–bgaH plus c-gvpE-pJAS) turned dark blue, demonstrating the induction of mcA1–bgaH expression by the cGvpE protein (mcA1/cE, Fig. 2). The cA1–bgaH 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 cA1–bgaH/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 pA1–bgaH construct (pA1) remained red although the p-gvpA gene is transcribed in p-gvpA transformants. Since the cA1–bgaH and pA1–bgaH 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.



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Fig. 2. Colonies of Hf. volcanii transformants sprayed with X-Gal. The cells were grown as cross-shaped colonies on agar medium and sprayed with 10 mg X-Gal ml-1. The constructs present in each transformant are indicated above the colonies. The designation ‘bgaH’ indicates the presence of plasmid pMLH32, and ‘cE’ indicates construct c-gvpE-pJAS. The respective A1–bgaH and A–bgaH fusion genes are inserted in pWL102. The origin of the promoter in the A1– and A–bgaH fusion genes is indicated by ‘mc’ (mediterranei chromosomal), ‘c’ (c-vac) and ‘p’ (p-vac).

 
Northern analyses were performed to monitor the A1–bgaH transcriptional levels. Total RNA was isolated from the transformants, electrophoretically separated on an agarose gel and hybridized with a bgaH-specific probe (Fig. 3). The 2·8 kb bgaH mRNA (and degradation products) was detected in pA1–bgaH and mcA1–bgaH transformants, whereas cA1–bgaH transformants did not contain detectable amounts of bgaH transcript. These results correlated with the earlier mRNA studies on the respective gvpA transformants. The mcA1–bgaH/cE and cA1–bgaH/cE transformants contained large amounts of bgaH mRNA, demonstrating that the transcription of both A1–bgaH genes was activated by cGvpE. Since the transcript formation in all of these transformants was as expected, the lack of ß-galactosidase activity in case of the pA1 and cA1/cE transformants was likely due to a problem that occurred at the level of translation.



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Fig. 3. Northern analysis to determine the presence of bgaH transcripts. RNA samples derived from exponential growth of the pA1–, mcA1– and cA1–bgaH, and mcA1–bgaH/cE and cA1–bgaH/cE transformants were separated on 1·2% agarose gels and hybridized with the 2·2 kb NcoI–BamHI bgaH fragment as strand-specific probe. The RNA marker sizes (in kb) are given on the left. ß-Galactosidase activity was also determined as indicated below the autoradiogram: -, (+) and + indicate the intensity of the blue colour (see Fig. 2).

 
Reporter gene fusions within the gvpA reading frame
The second series of fusion genes (A–bgaH) contained the bgaH reading frame fused to the larger pA, cA or mcA promoter fragments. The fusion was generated in the gvpA reading frame, resulting in ß-galactosidase proteins containing five amino acids of GvpA (MAQPD in the case of the p-gvpA and c-gvpA, and MVQPD in the case of the mc-gvpA fusion). Transformants contained either the A–bgaH construct by itself (to determine the basal product levels), or together with one of the three gvpE-pJAS constructs. All transformants were analysed by Southern blotting for the presence of the desired plasmids. Colonies of each of these transformants were sprayed with X-Gal solution for the detection of ß-galactosidase activity (Fig. 2, bottom). The mcA–bgaH transformants (mcA) were light blue, presumably due to low ß-galactosidase production, the cA–bgaH transformants (cA) remained orange-red (no ß-galactosidase production), and the pA–bgaH transformants (pA) were blue due to a higher amount of ß-galactosidase activity. Each transformant containing one of the A–bgaH genes plus c-gvpE-pJAS showed a significant increase in blue colour: the activation was strongest with the mcA–bgaH/cE transformant (mcA/cE), but also the cA–bgaH/cE (cA/cE) and the pA–bgaH/cE transformants (pA/cE) turned dark blue (Fig. 2, bottom). Thus, the A–bgaH fusion genes revealed ß-galactosidase activities according to the gvpA mRNA levels in the respective gvpA+c-gvpE (or +mc-gvpE) transformants (Röder & Pfeifer, 1996 ; Krüger et al., 1998 ).

The expression of the different A–bgaH 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 mcA–bgaH 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 A–bgaH genes was found with pA–bgaH transformants; mcA–bgaH transformants showed a low activity, whereas cA–bgaH transformants did not contain detectable ß-galactosidase activity (Table 2). The activation of each A–bgaH gene by GvpE was tested in the three different A–bgaH/E transformants. The various mcA–bgaH/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 pA–bgaH gene was also activated by all three GvpE proteins, but the expression of cA–bgaH was only achieved with the homologous cGvpE protein (cA–bgaH/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 pA–bgaH transformants. None of the heterologous GvpE activator proteins was able to activate the cA–bgaH gene (Table 2). In each case, the cGvpE protein appeared to be the strongest transcriptional activator protein among the three GvpE proteins (Table 2).



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Fig. 4. Growth curves and ß-galactosidase activities determined for mcA–bgaH and various mcA–bgaH/E transformants. Each plot shows the growth curve ({bullet}) and the specific ß-galactosidase activity ({triangleup}) of transformants containing the mcA–bgaH gene (mcA), or mcA plus mc-gvpE (mcE), c-gvpE (cE) or p-gvpE (pE) in pJAS35. ß-Galactosidase activities were determined by the ONPG assay.

 

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Table 2. Specific ß-galactosidase activities of the various transformants

 
Amount of bgaH mRNA and ß-galactosidase activity throughout growth
The different A–bgaH and respective A–bgaH/cE transformants were investigated at the mRNA level for the expression of the A–bgaH gene. The cells were grown in 25% salt medium, and samples were taken during the exponential and stationary phases for RNA isolation and Northern analysis. The 2·8 kb bgaH mRNA (and degradation products) was detected in all transformants (data not shown). The mcA–bgaH/cE transformant was used to compare the amount of bgaH mRNA produced during growth with the ß-galactosidase activities measured in the respective cell lysates. Eleven samples were taken throughout growth in 25% salt medium, and RNA was isolated and hybridized with the bgaH-specific probe (Fig. 5). The bgaH mRNA was mainly detected in samples derived from exponential and early stationary phases (Fig. 5b, lanes 1–7). Only minimal amounts of bgaH mRNA were detectable after 185 h (Fig. 5b, lanes 8–11). The ß-galactosidase activity was also determined for each sample and indicated an increase during the exponential and the early stationary phase (up to 150 h), which paralleled the increase in the bgaH mRNA (Fig. 5a). However, despite the minor amounts of bgaH mRNA observed during the stationary phase, the ß-galactosidase activities remained constantly high, indicating that the ß-galactosidase protein was rather stable and remained active.



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Fig. 5. (a) Growth curve and ß-galactosidase activities, and (b) Northern analysis to detect bgaH mRNA in the mcA–bgaH/cE transformant. (a) Samples were taken during growth ({bullet}), and the specific ß-galactosidase activities ({triangleup}) determined by the ONPG assay. The numbers above the growth curve indicate samples used for Northern analysis. (b) RNA isolated from samples 1–11 was used for Northern analysis and hybridized with a digoxigenin-labelled, bgaH-specific probe. RNA marker sizes (in kb) are given on the right.

 
pAcA promoter to investigate the region required for GvpE activation
A chimeric gvpA promoter was constructed by substituting a region of 21 nt 5' to the TATA box of the cA promoter with the respective 21 nt derived from the pA promoter (see Fig. 1a) and the resulting pAcA–bgaH gene was tested for ß-galactosidase activities and possible activation by the various GvpE proteins in transformants. Transformants containing the pAcA–bgaH construct did not produce detectable amounts of ß-galactosidase activity, similar to transformants containing the cA–bgaH construct (Table 2). However, the transformants harbouring the pAcA–bgaH construct plus one of the three gvpE-pJAS constructs produced ß-galactosidase, with pAcA–bgaH/pE exhibiting the lowest and pAcA–bgaH/cE the highest ß-galactosidase activity (Table 2). Compared to the transformants containing the original pA- and cA–bgaH sequences, a threefold higher ß-galactosidase activity was found compared to cA–bgaH/cE, but the pAcA–bgaH/cE transformant attained only 36% of the ß-galactosidase activity of the pA–bgaH/cE transformant (Table 2). Thus, the pAcA promoter acquired the ability for activation by the heterologous GvpE activator proteins.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The promoter of the plasmid-encoded p-gvpA gene of Hb. salinarum PHH1 is highly active, whereas the respective c-gvpA and mc-gvpA promoters depend on the activation mediated by the GvpE activator proteins (Röder & Pfeifer, 1996 ; Krüger et al., 1998 ). The bgaH reading frame encoding the ß-galactosidase of ‘Hf. alicantei’ (Holmes & Dyall-Smith, 2000 ) was used to investigate the basal and induced activities of the various gvpA promoter regions in Hf. volcanii transformants. Different fusion sites between the promoter fragments and bgaH were tested. One series of constructs contained the bgaH reading frame fused directly at the ATG start codon of the respective gvpA gene (pA1–, cA1– and mcA1–bgaH), whereas the second series (pA–, cA– and mcA–bgaH) contained the fusion site further downstream, resulting in ß-galactosidase proteins containing five additional amino acids of GvpA at the N terminus.

Reporter gene fusions at the ATG start codon of the gvpA gene may lead to problems in translation
All A1–bgaH and A1–bgaH/cE transformants contained the expected amount of bgaH mRNA. The mcA1–bgaH, cA1–bgaH and mcA1–bgaH/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 pA1–bgaH and cA1–bgaH/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 A->C) 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 cA1–bgaH mRNAs. However, a similar alteration was introduced in the mcA1–bgaH 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 stem–loop 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 mcA–bgaH constructs revealed the expected mRNAs that, when present, were translated into ß-galactosidase. The analysis of the product formation in each A–bgaH transformant indicated no mRNA and ß-galactosidase in the cA–bgaH transformant, a low mRNA amount and ß-galactosidase activity in the mcA–bgaH, and a high mRNA amount and ß-galactosidase activity in the pA–bgaH 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 A–bgaH 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 mcA–bgaH 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 pA–bgaH construct was also stimulated by all three GvpE proteins in respective pA–bgaH/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 mcA–bgaH and MAQPD in the case of cA– and pA–bgaH). However, the products of the pA–bgaH and the cA–bgaH genes are identical, and at least these specific ß-galactosidase activities can be compared with each other.

Similar to the c-gvpA gene, the cA–bgaH 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 pAcA–bgaH 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 pAcA–bgaH 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 pAcA–bgaH 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 mcA–bgaH/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 promoter–bgaH 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
 
This work received financial support from the Deutsche Forschungsgemeinschaft (PF 165/6-3 and 165/8-1). We thank Mike Dyall-Smith for plasmid pMLH32 prior to publication, Richard Röder for plasmid mc-gvpE-pJAS, and Peter Zimmermann, Jobst Gmeiner and Kathryn Nixdorff for critical reading of this manuscript. Lovastatin was a generous gift of MSD Sharp & Dohme GmbH (München).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 18 January 2001; revised 15 March 2001; accepted 19 March 2001.