This gene was cloned by M. Holmes, in my lab, from Hfx. lucentense (previously "Hfx. alicantei"), and has been successfully used by us and others (see references below). The gene was made available to halobacterial labs in 1996, 4 years before we published the sequence. The basic enzymology came out in BBA in 1997. A description of the gene, its sequence and its expression came out in 2000 (Mol Microbiol), back-to-back with a paper from Nadja Patenge and colleagues (Oesterhelt's lab at the MPI). They used bgaH to measure promoter activity in Hbt. salinarum. I made contact with Nadia some time previously and we submitted our manuscripts together. It was a great pleasure when they both came out! Why the delay of 4 years?? Grant reviewers in Australia thought it rather boring stuff ('just E.coli all over again'), and our Research Council (ARC) would not fund it.
Despite the ARC, we continued to improve the system, including better vectors (' you 'aint seen nothin yet! '). It has been a crucial tool in our studies of gene expression in haloviruses. The bgaH gene is now used around the world. ONPG, X-Gal and fluorescent substrates can be used as easily as with E.coli LacZ. The enzyme is a member of glycohydrolase family 42, a completely different group to that of E.coli LacZ, and, surprisingly, is almost certainly a homo-trimeric enzyme, based on the crystal structure of a closely related enzyme from Thermus sp. (Hidaka et al., 2002). This makes BgaH a biologically novel type of enzyme, in terms of its structure, halophilicity, and its as yet unkown natural substrate.
References are at the bottom of this page. The Halohandbook, from this site, gives detailed methods for assay of enzyme activity. The nucleotide sequence accession U70664 (includes bgaH and related genes in a 5.4 kb genomic clone). The protein sequence accession is AAB40123 (BgaH only).
Useful information for those using bgaH as a reporter
- The transcription start point (at least the major one) of wt bgaH (as in pVA513 or pMLH32 )is -34 from A of AUG start codon
- The mRNA is (apparently) 2.1 kb, ie about the right length for the ORF. In
Hfx. volcanii you may see a shorter transcript as well as the full-length one. This was not seen by Nadja Patenge et al. when she used the gene in
Hbt. salinarum. When we analysed this more recently (2000), we couldn't see the shorter transcript either, so we think it is an artefact of the mRNA preparation, or perhaps a natural degradation product. The half-life of the mRNA is short (but the protein half-life is long; as seen by Gregor and Pfeifer (2001).
- Transformation into Hf. volcanii (try a wt isolate as well as WFD11) with pMLH32 gives a specific activity of about 0.4 - 0.5 U/mg. (see the beta-galactosidase assay method listed in the Halohandbook protocol manual).
- Use the radA strain of Hfx. volcanii for greater stability (strain DS52 from this lab). It grows more slowly but it is worth it.
- Activity of the gene in the wt strain is greatly increased when the strain is grown on galactose, and is least when grown in rich media (peptone/yeast extract). With the cloned (mutant) gene, when present in Hfx. volcanii cells, activity is high in most media, rich or not. I think this is due to the plasmid (pMLH32) having some outside transcription from E.coli plasmid sequences. We have now blocked this in our recent vectors (pRV1; B. Russ, 2006, unpublished).
- 1. Don't change or delete any of the initial codons of bgaH: it looks to be very sensitive to changes at that end.
- 2. Changing the start codon region to an NdeI site is OK, but to an NcoI site (which changes bases downstream) is not !
- 3. The host can eliminate plasmid based beta-gal expression if it is too high. Use a radA host strain (eg. DS52) to prevent this when studying strong promoters. (For the same reason, the E.coli people use recA hosts.)
- 4. Watch out for salt interference with protein assays when you are determining specific activities.
- 5. You can trim down the 3' and 5' ends of the pMLH32 clone of bgaH (not the ORF!) without affecting expression and activity.
- 6. You can use different promoters easily via an NdeI site at the start codon (we have a vector for this, pRV1, and one with a ClaI site, pRV2).
- 7. NOTE: "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 beta -galactosidase" (Gregor and Pfeifer, 2001). We find that 5aa or more of the foreign gene (as a translational fusion to BgaH) is enough to overcome the problem.
- 8. What is the natural substrate for BgaH ? We don't know. Nor does anyone else who studies family 42 glycohydrolases (the family that bgaH belongs to). However, we now know you can use phenyl-galactoside (P-Gal) as a growth substrate for Hfx. volcanii cells that contain bgaH (discovered by M. Rizkalla, 2000). (email me for details)
- Large, A., Stamme, C., Lange, C., Duan, Z., Allers, T., Soppa, J. and Lund, P.A., 2007. Characterization of a tightly controlled promoter of the halophilic archaeon Haloferax volcanii and its use in the analysis of the essential cct1 gene. Molecular Microbiology, 66(5), 1092-1106.
- Zhu, J.Y., Hu, Y.H., Liu, J., Huang, Y.P., and Shen, P. (2006) [Analysis of promoter sequence and its activity of homologues gene rad25 of eukaryotes from halophilic archaea]. Wei Sheng Wu Xue Bao 46: 196-199.
- Gregor, D., and Pfeifer, F. (2005) In vivo analyses of constitutive and regulated promoters in halophilic archaea. Microbiology 151: 25-33.
- Hofacker, A., Schmitz, K.M., Cichonczyk, A., Sartorius-Neef, S., and Pfeifer, F. (2004) GvpE- and GvpD-mediated transcription regulation of the p-gvp genes encoding gas vesicles in Halobacterium salinarum. Microbiology 150: 1829-1838.
- Sartorius-Neef, S., and Pfeifer, F. (2004) In vivo studies on putative Shine-Dalgarno sequences of the halophilic archaeon Halobacterium salinarum. Mol Microbiol 51: 579-588.
- Zimmermann, P. and Pfeifer, F.(2003) Regulation of the expression of gas vesicle genes in Haloferax mediterranei: interaction of the two regulatory proteins GvpD and GvpE. Mol Microbiol. 49(3):783-94.
- Gregor, D. and F. Pfeifer (2001). Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea. Microbiology 147(Part 7): 1745-1754.
- Holmes, M. L. and M. L. Dyall-Smith (2000). Sequence and expression of a halobacterial beta-galactosidase gene. Molecular Microbiology 36(1): 114-122.
- Patenge, N., A. Haase, et al. (2000). The gene for a halophilic beta-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum. Molecular Microbiology 36(1): 105-113.
- Holmes, M. L., R. K. Scopes, et al. (1997). Purification and analysis of an extremely halophilic beta-galactosidase from Haloferax alicantei. Biochim Biophys Acta 1337(2): 276-86