Transcriptionally active regions in the genome of the archaebacterium Haloferax volcanii

Abstract

TranscriptionaUly active regions of the Haloferax vokcanii genome were mapped by hybridization of radiolabeled cDNA to Southern blots of our minimal set of overlapping cosmid clones covering 96% of the 4.1-Mbp genome. Transcription during exponential growth occurred in nearly every region of the 2,920-kbp chromosome. Large parts of the 690-and the 86-kbp plasmids were transcribed, but the 440-kbp plasmid showed little expression. Transcription after a 40-min heat shock at 65°C was generally reduced, apart from a smal set of strongly expressed loci all situated on the chromosome.A high-resolution physical map of the 4.1-Mbp genome of the archaebacterium Haloferax volcanii DS2 has been recently completed (3). The genome is partitioned into five circular replicons: a chromosome of 2,920 kbp and four plasmids of 690, 440, 86, and 6.4 kbp. The detailed study of halobacterial genome structure is useful in that it provides an archaebacterial data base for comparative genome analysis, and it addresses the issue of the dynamics and evolution of a genome rich in insertion elements (4,8,15,16) and plasmids.We and our collaborators are interested in mapping genes to the physical framework of overlapping cosmid clones which currently cover 96% of the H. volcanii genome. The following three approaches have been used: (i) hybridization of Southern blots of digested cosmid clones by using bulk probes (tRNA or insertion elements) has allowed the mapping of families of genes (4), (ii) individual cloned halobacterial genes have been mapped by homologous or heterologous hybridization (3), and (iii) complementation of auxotrophic mutants with cosmid clone DNA has mapped 140 mutations to 35 loci (4,5,10).With the goal of further understanding the genome of H. volcanii, we designed an experiment to find those loci most transcriptionally active under different physiological conditions by synthesizing radiolabeled cDNA and hybridizing it to Southern blots of digests of our minimal set of overlapping cosmid clones. This report describes the transcriptional organization of the genome of a culture in exponential growth (in chemically defined medium [CDM] and in complex medium) and compares it with that of a culture experiencing heat shock. MATERIALS AND METHODSGrowth media and conditions. CDM was a modification of that described by Kauri et al. (9) in which glycerol and succinate were replaced with 30 mM trisodium citrate. Rich medium was as described in reference 6. Cultures (50 ml) were grown at 45°C with shaking in 250-ml Erlenmeyer flasks. Cells were harvested during exponential growth (A540, 1.6) in rich medium, during exponential growth (A540, * Corresponding author.1.0) in CDM, or after exponential growth (A540, 1.0) in CDM followed by a 40-min heat shock at 65°C with occasional agitation.RNA extraction. Cells were quick-chilled to -15°C by immersing and swirling the flask for 2 min in a salted ice-water bath before the culture was centrifuged at -10 to -15°C for 10 min at 6,000 x g. The cell pellet was suspended in 0.25 ml of col...

Differentially transcribed regions of Haloferax volcanii genome depending on the medium salinity

“…In a large domain (around positions 1000 to 1500 in the chromosome), although it is quite transcriptionally active under optimal conditions and is also widely involved in heat shock response (21), salinity does not seem to trigger significant differential expression (data not shown). The 442-kb megaplasmid pHV3 was shown to be transcriptionally rather inactive, while the 86-kb pHV1 plasmid appeared to be quite active (21). Accordingly, we have in fact detected little response to salt in pHV3 and found a significant salt modulation in the smaller pHV1 plasmid (data not shown).…”

“…2) with remarkable enhancement of expression under low-salt conditions. This is one of the sequences showing the clearest responses to salt, but the same 2.2-kb MluI fragment was described as having a strong heat shock response and also as being induced in minimal medium (21), so it would probably be more aptly considered as a sequence related to general stress.…”

“…The region from clones 437 to 470 shows, as mentioned above, enhancement by high-salt conditions, although it is not very apparent in most cases. Heat shock induction specifically within these two cosmids has been described previously (21).…”

“…In fact the organism, like other Haloferax species (9,17), presents a broad and flat growth response, with no significant changes in growth rates from about 12 to 30% salts in the media used here. H. volcanii DS2 was grown in complex medium containing either 12, 20, or 30% of a mixture of salts (SW) in the proportions found in seawater as described previously (18) The technique involves hybridization of cDNA probes obtained at different conditions against the clones of an ordered library of the organism (4,21). A collection of overlapping cosmid clones covering most of the H. volcanii genome has been developed (2) and was kindly provided to us by W. F. Doolittle.…”

“…32 P]dCTP to the newly synthesizing cDNA strand by using random hexamer primers and Moloney murine leukemia virus reverse transcriptase), as described previously (20,21), from total RNA samples extracted from cells grown at the different salt concentrations in the logarithmic phase.…”