The intracellular location of methane mono-oxygenase (MMO) (soluble or particulate) in Methylosinus trichosporium OB3b is dependent on the availability of copper in the growth medium. Raising the Cuz+ concentration from 1 VM to 5 p~ effected a transition from soluble to particulate MMO activity, and changes in major cell polypeptides were observed on SDSpolyacrylamide gels. Organisms containing soluble MMO oxidized a wide range of substrates including n-alkanes, n-alkenes, aromatic and alicyclic compounds. By contrast, organisms containing particulate MMO did not oxidize aromatic or alicyclic compounds. These observations provide further evidence that the two types of MMO are fundamentally different.

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...

Extracellular lipase was purified from a Tween 80-limited continuous culture of Pseudomonas aeruginosa EF2 by ultrafiltration of the culture supernatant followed by anion-exchange and gel-filtration FPLC. The lipase was composed of a single subunit (M, 29000, PI 4.9), which was capable of a variable degree of aggregation, and which exhibited both lipase activity, measured with the insoluble substrate olive oil (predominantly triolein), and esterase activity, measured with the soluble substrates p-nitrophenyl acetate and Tween 80. Lipase activity was approximately eight times higher than either type of esterase activity (kcat approximately 3OoOs-' for the hydrolysis of olive oil). The enzyme showed a marked regiospecificity for the 1,3-oleyl residues of radiolabelled triolein, was relatively stable at moderate temperatures (exhibiting a biphasic loss of activity with an initial fi of 17.5 min at 60 "C) and was very stable to freezing and thawing. Lipase activity was only weakly inhibited by the serine-active reagent 3,4-dichloroisocoumarin, and was not inhibited by the chelating agent EDTA (1 mM). The Nterminal amino acid sequence of the Ps. aeruginosa EF2 lipase showed a marked similarity to those of several other bacterial lipases.

~ ~~ ~~ ~ ~ In cicv 13C NMR has been used to observe metabolism of exogenously supplied methanol by suspensions of Methylosinus trichosporium OB3b grown under a variety of conditions. Formaldehyde, formate and bicarbonate ions were the only metabolites of methanol to be detected. Accumulation of formaldehyde was observed only with suspensions grown under conditions which yield particulate, membrane-bound, methane mono-oxygenase (MMO). Ethyne abolished MMO activity, partially inhibited methanol oxidation in whole organisms, and prevented growth of the organism on methanol ( 1 %, v/v) in batch culture. Oxidation of ethanol, a substrate of methanol dehydrogenase, was not affected by ethyne. Ethyne caused accumulation of formaldehyde in all suspensions of the organism incubated with methanol, although oxidation of exogenously added formaldehyde was not affected. These observations are consistent with the proposal that in M. frichosporium OB3b both MMO and methanol dehydrogenase oxidize exogenously supplied methanol and suggest that the further oxidation of formaldehyde is stimulated by the consumption of reducing equivalents by MMO.

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...