Gene, 110 (1992) 9-16 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05,00 GENE 06271

Cloning and characterization of a gene from Bacillus stearothermophilus var. non-diastaticus encoding a glycerol d e h y d r o g e n a s e (Recombinant DNA; thermophile; kinase; Zym~monas; Clostridium)

Philip R. Mallinder*, Andrew Pritchard and Anne Moir Department of Molecular Biology and Biotechnology, The Krebs Institute, University of Sheffield, Sheffield, SIO 2TN (U.K.) Received by K.F. Chater: 3 May 1991 Revised/Accepted: 23 September/25 September 1991 Received at publishers: 7 November 1991

SUMMARY

A 4.1-kb EcoRI fragment which includes the gene (gldA) encoding a glycerol dehydrogenase (GIDH; EC 1.1.1.6; glycerol:NAD oxidoreductase) from Bacillus stearothermophilus var. non-diastaticus has been cloned by virtue of its ability to restore glycerol utilisation to Escherichia coli glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD) mutants. Sequencing suggests that the gldA gene is likely to be monocistronic and encodes a protein of 39450 Da. The deduced amino acid composition and sequence of GIDH reveals that the protein is extremely similar to a characterized metal-dependent NAD-dependent GIDH from B. stearothermophilus RS93. The enzyme has limited homology to the ironactivated alcohol dehydrogenase of Zymomonas mobilis and the butanol dehydrogenase of Clostridium acetobutylicum.

INTRODUCTION

Many prokaryotes utilise glycerol as a source of carbon and energy. Glycerol dissimilation can occur by two routes: one involves GIK-mediated phosphorylation to G3P, followed by dehydrogenation to dihydroxyacetone phosphate, whereas in the second route GIDH acts to produce DHA, which is then phosphorylated. Glycerol dissimilation and its regulation have been reviewed by Lin (1976). In E. coil, glycerol enters the cell by facilitated diffusion (Richey and Lin, 1972), and a tetrameric GIK of 55-kDa Correspondence to: Dr. A. Moir, Department of Molecular Biology and Biotechnology, University of Sheffield, P.O. Box 594, Sheffield S 10 2UH (U.K.) Tel. (44-742)768555; Fax (44-742)728697. * Current address: Department of Biochemistry, ICI Joint Laboratory, University of Leicester,LeicesterLE! 7RH (U.K.) Tel. (44-533)522522. Abbreviations: A, absorbance; aa, amino acid(s); Ap, ampicillin; B., Bacillus; bp, base pair(s); DHA, dihydroxyacetone; DHAP, DHA phos-

subunits catalyses the formation of G3P from glycerol and ATP (Thomer and Paulus, 1971). G3P is oxidised via an FAD-dependent, membrane-associated, G3P dehydrogenase to DHAP; separate enzymes function under aerobic and anaerobic conditions (Kistler and Lin, 1972; Schryvers et al., 1978). Kiebsiella aerogenes 1033 possesses all the dissimilatory enzymes of E. coli as well as an NAD-dependent G1DH and a DHA kinase. The DHA pathway (oxidation by GIDH followed by phosphorylation by DHA kinase) is used during fermentative dissimilation of glycerol and the phate; FAD, flavin adenine dinucleotide; GIDH, glycerol dehydrogenase; GIK, glycerol kinase; gldA, gene encoding GIDH; glpD, gene encoding glycerol-3-phosphate dehydrogenase; glpK, gene encoding GIK; G3P, glycerol-3-phosphate; IPTG, isopropyl-fl-D-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; M9, minimal salts (medium) (see Maniatis et al., 1982); nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; pfu, plaque-forming units; R, resistance/ resistant; SDS, sodium dodecyl sulfate; XGal, 5-bromo-4-chloro-3indolyl-fl-D-galactopyranoside; [ ], denotes plasmid-carrier state.

10 G3P pathway (analogous to that of E. coli) is used during oxidative dissimilation of glycerol (Ruch et al., 1974). The main pathway for glycerol dissimilation in B. subtilis is analogous to that of E. coil, operating via a G1K and NAD-dependent G3P dehydrogenase; the genes concerned have been mapped, cloned and partially sequenced (Holmberg et al., 1990). Several strains of B. stearothermophilus are capable of growth on glycerol as sole carbon and energy source (Atkinson et al., 1975; Spencer et al., 1989; Burke and Tempest, 1990). The route of glycerol dissimilation in B. stearothermophilus has not been determined but may be strainspecific due to the extreme heterogeneity of the species designation. Atkinson et al. (1975) reported the presence of GIK in extracts of glycerol-grown B. stearosthermophilus NCA1503, and Burke and Tempest (1990) reported very high activities of GIK in extracts of B. stearothermophilus var. non-diastaticus grown in glycerol-limite,'d chemostat culture. The alternative metabolic route may also operate; Spencer et al. (1989) purified a GIDH from B. stearothermophilus RS93, a strain which is closely related to B. stearothermophilus var. non-diastaticus (Sharp et al., 1989). This enzyme would be more strictly named giycerol:NAD oxidoreduetase, as an in vivo role in glycerol catabolism has not been established, but GIDH, the name already used in the literature (Spencer et al., 1989; 1990), will be used here. A phage 2 library containing B. stearothermophilus var. non-diastaticus DNA was used to restore the ability to grow on glycerol as sole carbon source to E. coli mutants defective in glycerol dissimilation. As the gene cloned proved not to encode a GIK, the process did not represent true complementation, but the term is used in the looser, more general sense in the rest of the paper. The 'complementing' gene, encoding GIDH, was recovered from a 2 transducing phage; its sequence and gene organisation are reported.

RESULTS AND DISCUSSION

(a) Complementation of Escherichia coli glpK and glpD mutants A gene library of B. stearothermophilus chromosomal DNA was prepared in 2ZAPII. An EcoRl partial digest (3/~g) was mixed with a complete EcoRl digest (1 #g) and fragments in the size range 2-9 kb (20-50 ng) were ligated with 200 ng of EcoRI-linearised 2ZAPII DNA (Stratagene, La Jolla, CA; the vector cos sites had been ligated before digestion), packaged into 2 phage heads using Gigapack II Plus packaging extract (Stratagene) and amplified according to the manufacturer's instructions on the E. coli host XLI-Blue (endA 1 hsdR 17(r£ rnff:)supE44 thi-I 2- recA gyrA96 relA 1 lac [F' proAB + laclQZAM15 Tnl0]; provided by Stratagene).

An attempt was made to complement E. coli glycerol metabolic defects with the gene library: cultures of E. coil strains Lin61 (F + glpK tonA 22 lac-20 ompF627 relA 1 pit10 spoT1 T2R; provided by B. Bachmann) and Lin95 (F + glpD tonA22 phoA8 ompF627 fadL701 relA 1 pit-lO spoT1 T2R; also from B. Bachmann) were grown according to Cozzarelli et al. (1968) to A600= 0.7, in M9 minimal salts medium (Maniatis et al., 1982) supplemented with 1~o (w/v) Casamino acids/0.5% (w/v) glucose/0.5% (w/v) maltose, then harvested and resuspended in 10mM MgSO4. Dilutions of the amplified library (105-107 pfu) were mixed with 200 #1 of cells. After adsorption at room temperature for 15 min, top agar in M9 minimal salts) was added, the mixture plated onto M9 agar containing 0.2% (v/v) glycerol as carbon source, and the plates incubated for ten days at 30°C. The detection of 'complementing' lysogens was complicated by the significant reversion of the gip hosts, as observed on control plates. Twenty to thirty colonies, which could represent revertants and/or lysogens carrying a complementing prophage, were purified to single colonies on M9 glycerol plates. The 2c1857 prophages were heat-induced from 5 mi cultures of lysogens grown at 30°C in L broth to A60o---0.7. After 5 min at 46°C cultures were shaken at 370C for 2 h, cell debris was removed by centrifugation and the lysate was treated with DNase (1 #g/ml final concentration), then sterilised by shaking with a drop of chloroform. The lysates were then diluted 100-fold in phage buffer and 10 #1 used in spot transductions on selective plates as used in the initial complementation. Colonies were visible after five to six days incubation at 30°C, but grew relatively slowly. One of the lysates, originally isolated from a glpD complementation experiment, complemented the glpK defect in spot tests, This hybrid phage, designated 95.5, restored growth to the E. coil glpK mutant and to a lesser degree to the glpD mutant. DNA from phage 95.5, which contained a single cloned EcoRl fragment of approx. 4.0 kb in the cloning site of the phagemid within 2ZAPII, was obtained using the method described by Errington (1984). This 4-kb fragment was excised in vivo from the phage genome and packaged in vivo using the helper phage R408, according to the standard protocol supplied by Stratagene. The virions produced were used to infect XL1-Blue, Lin61 or Lin95, selecting for Ap a colonies, These were purified before screening for growth on glycerol. Lin61 (gipK) cells carrying the phagemid from 95.5 (designated pPRM1) grew on glycerol minimal medium. The gipD mutants were partially complemented by pPRM1, growing to small colonies. The restriction map of the cloned region is shown in Fig. 1; regions from pPRM1 were subcloned in pUC119 to yield pPRM2 and pPRM3. Southernblot analysis confirmed the origin of the cloned DNA, as

lkb

I

l

E

H l

pPRM1

ORF1

I

Bg l

ORF2 39]rJDa

H

Bc

Ii

H

ORP3

S

E

I

I ORF5

I

I ORF4

pPRM2

I

pPl~3

I

I

I

Fig, 1. Organisation of ORFs within the cloned DNA. The restriction map of the cloned 4161-bp EcoRl fragment in pPRMI is shown, and the positions and orientations (arrows) of ORFs deduced from the nt sequence are indicated. The vector portion of pPRM 1 is pBluescript SK -. Plasmid pPRM2 was produced by ligating the 2.7-kb Smal fragment from pPRM 1 - one of the Sinai sites is in the polylinker preceding the cloned fragment - with Sma.digested pUC119 DNA. Plasmid pPRM3 was derived from the 2-kb HindIII-EcoRI fragment of pPRM1, cloned between the HindIII and EcoRI sites of pUCII9. Bc, Bcil; Bg, Bgll; E, EcoRi; H, HindIII, S, Smal.

it hybridises to an identically sized fragment in an EcoRI digest of B. stearothermophilus var. non-diastaticus DNA. (b) Enzyme assays To determine the nature of the complementing activity, GIK and GIDH assays were carried out on extracts of E. coil strains carrying each of the plasmids (Table I). Plasmids pPRM 1 and pPRM2 conferred significant GIDH activity, but not GIK activity. A gidd gene therefore lies within the 2.7-kb pPRM2 subclone. This observation led to a reassessment of the report of Burke and Tempest (1990) who, using different assay conditions, failed to detect GIDH activity in this B. stearothermophilus strain. Extracts of chemostat-grown glycerol-limited cells of B. stearothermophilus did in fact contain GIDH activity at significant levels (Table I), at a specific activity at least 40-fold higher than that reported by Spencer et ai. (1989) for batch-grown cells of B. stearothermophilus RS93. GIK assay of the same cell extract (Table I) suggested that the kinase and dehydrogenase activities, at least when measured at sub-optimal temperatures of 37°C and 40°C, respectively, were of the same order of magnitude. Unlike the GIK, however, the GIDH could not be identified as a major band after SDSPAGE of total cell protein. (c) Identification of the gldA gene In vitro transcription and translation of plasmid DNAs are shown in Fig. 2. In addition to the//-lactamase (approx. 33 kDa) synthesised from each plasmid, both pPRM1 and pPRM2 encoded a protein of approx. 42 kDa that is not synthesized from pPRM3 or the vector.

TABLE I GIDH and GIK activities of plasmid-bearing strains of Escherichia coli and Bacillus stearothermophilus Strains a

E. coli K-12 W311OgipK + (wild-type) Lin61 glpK glpK[pBluescript] glpK[pPRMl] glpK[pPRMI]+IPTG glpK[pPRM2] glpK[pPRM2] + IPTG glpK[pPRM3] glpK[pPRM3] + IPTG

B. stearothermophUus var. non.diastaticus

GIDH b

GIK ~

40oc

52oc

37oc

Cloning and characterization of a gene from Bacillus stearothermophilus var. non-diastaticus encoding a glycerol dehydrogenase.

A 4.1-kb EcoRI fragment which includes the gene (gldA) encoding a glycerol dehydrogenase (G1DH; EC 1.1.1.6; glycerol:NAD oxidoreductase) from Bacillus...
848KB Sizes 0 Downloads 0 Views