The basal transcription apparatus of Archaea corresponds to the core machinery of the eucaryal RNA polymerase II system. However, it is not yet known how regulation of archaeal transcription is achieved. Examination of complete archaeal genome sequences reveals homologs of bacterial transcriptional regulators. We have studied one such molecule, MDR1, an A. fulgidus homolog of the bacterial metal-dependent transcriptional repressor, DtxR. We find that in vivo expression of the MDR1-containing operon is regulated by metal ion availability. In vitro analyses show that MDR1 recognizes three operator elements in its own promoter in a metal-dependent manner. MDR1 negatively regulates transcription of its own gene in a reconstituted in vitro system, not by abrogating the binding of TBP or TFB to the promoter but by preventing RNA polymerase recruitment.

A 6.5-kb Ec'cnR1 fragment of gcnomic DNA from a Rhiznhium sp. cloned into pLJC19 was able to endow Escherichin coli K-12 with the novel ability to grow at the expense of 2-chloropropionic acid. Subcloning showed that this property was a consequence of two dehalogenases encoded on a 2.2-kb Pstl fragment. Further subcloning of the PstI fragment led to two constructs that encoded, scparatcly, dehalogenase activity that acted stereospecifically on u-2-chloropropionic acid and L-2-chlompropionic acid, respectively. The genes encoding these two stereospecific dehalogenases have been sequenced and shown to be separated by 177 bp of non-coding DNA. Expression of the dehalogenase genes involved lhe vector promoter, suggesting that the anticipated Rhirohium sp. regulatory sequences were not functional in E.coli. Comparison of the deduced amino acid sequences of the two dehalogenases (1 8% identity) indicated that there was no obvious cvolutionary relationship between (hem. Nor was there any striking identity with any other 2-chlompropionic acid dehalogenase studicd so far.Keywords: dehalogenase; 2-chloropropionic acid ; gene cloning ; scqucncc coinparison; Rhizobium.Halogenated organic compounds iirc round widely throughout the biosphere and their microbial catabolism has been reviewed recently 11 -41. This process generally involves enzymccatalysed carbon-halogen bond cleavage and the dehalogenation rcactions have been classified into different typcs [l, 21. The hydrolytic dehalogenases act on haloacids or haloalkanes and whether these three dehalogenases wcrc encoded by neighbouring genes and to seek information on the two stereospecific enzymes, we set out to clone and analyse the RItizohiunz sp. ClPpH dehalogenase genes. The outcome of some of this work is described here.within-this group &era1 2-chloropropionic acid (CIPpH) dehalogenases have been studied. The enzymes that act on ClPpH can be stcreospecific for the D or I. form or can act equally on both isomers. The only organism so far reported to make all three forms of ClPpH dehalogenase is a Rhiznhium sp. 151. De-halogenase I was shown to be specific for L-CIPPH and also acted on dichloroacctic acid (C12AcH) but not on 2,2-dichloropropionic acid (C1,PpH) or monochloroacetic acid (CIAcH). Dehalogenase T I was non-specific, acting on D/I.-CIP~H, Cl,PpH, CI,AcH, and CIAcH. Dehalogenasc 111 was shown to act only on D-CIP~H and CIAcH, with no activity towards CI,PpH or C1,AcH. For each dehalogeniisc the lactate produced from ClPpH has the opposite stereochemical form to that of the substrate. Studics involving mutant strains deficient in some or all of these enzymes implied that the dehalogenase I and 111 genes were linked and coordinately regulated 161. To investigate MATERIALS AND METHODSBacterial strains, plasmids and growth conditions. The Esclrerichiu coli K-12 strain NMS22 [7] was used as host for plasmids pUC18 and pUC19 [XI. The Rhizohium sp. 191 was grown aerobically at 30°C in a mineral salts rncdium 1101 containing 10 mM n,r.-CIPpH or 20 mM C1,PpH supplem...

A 3-kb EcoRI fragment of genomic DNA from a Rhizobium sp. cloned into pUC19 endowed Escherichia coli K-12 with the ability to grow, albeit slowly, with 2-chloropropionic acid as substrate. The construct expressed weakly a gene that encoded a non-stereospecific 2-chloropropionic acid dehalogenase (dehalogenase 11; DehE). The dehE gene was not closely linked to the organism's other two dehalogenase genes, dehD and dehL. The derived amino acid sequence of DehE showed little identity with DehD or DehL, but there was significant identity to two other dehalogenases that act non-selectively on Z-chloropropionic acid. The fragment carried a truncated ORF upstream of dehE that was 51% identical to a positively acting regulatory protein, DehR, required for expression of a Pseudomonas putida dehalogenase gene. In its complete form this gene could encode the Rhizobium sp. dehalogenase-regulatory protein.DehE dehalogenated tribromoacetic acid completely, forming stoichiometric amounts of carbon monoxide and carbon dioxide as the other products.Keywords : dehalogenase; haloalkanoic acid ; CO formation ; sequence comparison ; trihaloacetic acid.A bacterium isolated from soil by elective culture on 2,2-dichloropropionic acid (C1,PpH) and identified as a Rhizobium sp. was found to produce three haloalkanoate dehalogenases [I]. Mutant analysis suggested that formation of the enzymes was controlled by a single regulator gene [2], but only dehalogenase I1 acted on CI,PpH [I]. All three enzymes acted on 2-chloropropionic acid (ClPpH), with dehalogenase I (DehL) being stereospecific for L-CIPPH, dehalogenase 111 (DehD) being stereospecific for D-CIPpH, and dehalogenase I1 (DehE) acting on both stereoisomers [ 11. Dehalogenases I and 111 collectively acted on monochloroacetic acid, dichloroacetic acid, 2-chlorobutyric acid and 2,3-dichloropropionic acid, with dehalogenase 11 acting on all of these compounds [l, 31. It was curious, therefore, that the organism had dehalogenases I and I11 when dehalogenase I1 on its own could act on all the identified substrates of the other two dehalogenases, and only dehalogenase I1 could utilize CI,PpH, on which the organism was isolated.A possible explanation of this phenomenon was that dehalogenase I1 had evolved from dehalogenases I and I11 and in so doing had gained the additional ability to act on C1,PpH. We have isolated and analysed the dehalogenase-11-encoding gene, dehE, and investigated the unusual ability of the enzyme to dehalogenate trihaloacetic acids [3]. MATERIALS AND METHODSBacterial strains, plasmids and growth conditions. The Escherichia coli K-12 strain NM522 [4] was used as host for plasmids pUCl8 and pUC19 [5] and E. coli strain BL21 (DE3)[6] was used for plasmid pT7-7 [7]. Cells were grown aerobically at 30°C in a mineral salts medium [8] containing 10 mM D,L-CIPPH and necessary growth factors, or in Luria-Bertani medium [9]. Ampicillin (100 pg/ml) and isopropyl thio-p-D-galactoside (IPTG) were incorporated as appropriate. Carbon sources and supplements were sterilised separa...

In March 1996, a survey of hydrothermal sites on the island of Montserrat was carried out. Six sites (Galway's Soufrière. Gages Upper and Lower Soufrières, Hot Water Pond, Hot River, and Tar River Soufrière) were mapped and sampled for chemical, ATP, and microbial analyses. The hydrothermal Soufrière sites on the slopes of the active Chances Peak volcano exhibited temperatures up to almost 100 degrees C and were generally either mildly acidic at pH 5-7 or strongly acidic at pH 1.5-3, but with some hot streams and pools of low redox potential at pH 7-8. Hot Water Pond sites, comprising a series of heated pools near the western shoreline of the island. were neutral and saline, consistent with subsurface heating of entrained seawater. Biological activity shown by ATP analyses was greatest in near-neutral pH samples and generally decreased as acidity increased. A variety of heterotrophic and chemolithotrophic thermophilic organisms were isolated or observed in enrichment cultures. Most of the bacteria that were obtained in pure culture were familiar acidophiles and neutrophiles, but novel, iron-oxidizing species of Sulfobacillus were revealed. These species included the first mesophilic iron-oxidizing Sulfobacillus strains to be isolated and a strain with a higher maximum growth temperature (65 degrees C) than the previously described moderately thermophilic Sulfobacillus species.

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