Cloning and analysis of the β-galactosidase-encoding gene from Clostridium thermosulfurogenes EM1

Gene, 106 (1991) 13-19 Q 1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

13

0378-I 119/91/$03.50

06071

Cloning and analysis of the jbgalactosidase-encoding (Recombinant

DNA;

nucleotide

sequence;

sequence

comparison;

gene from Clostridium thermostable;

EM1

thermosulfurogenes

anaerobic)

Gerhard Burchhardt * and Hubert Bahl Institutftir Mikrobiologie, Georg-August-Universitiit.W-3400 Giittingen(F.R. G.) Received by J.-P. Lecocq: 27 November 1990 Revised/Accepted: 13 March/l9 July 1991 Received at publishers: 30 July 1991

SUMMARY

Clostridium thermosulfurogenes EM 1 produced a thermostable (up to 70 “C) B-galactosidase @Gal) with a pH optimum of 7 during growth on lactose. The gene (IacZ) encoding this enzyme was cloned and expressed in Escherichia coli using pUC18 as a vector. The nucleotide sequence of a 2.7-kb PstI fragment carrying the IacZ gene was determined. The open reading frame for facZ, which encoded a protein of 7 16 amino acids with a calculated M, of 83 728, was confirmed by the identity of its deduced aa sequence with the chemically determined N-terminal aa sequence of the purified /IGal of C. thermosulfurogenes EMl. The structural gene was preceded by a possible promoter sequence, 5’-TTGTAG (-35) 5’-TAATAT (-10); and a ribosome-binding site, 5’-AGGAGG. The cloned /IGal was found to be indistinguishable from the native enzyme. The A4, of the active BGal was 170000, as determined by Superose 12HR gel filtration and gradient gel electrophoresis. This indicated that this enzyme is composed of two identical subunits. Comparison of the aa sequences of different BGal revealed that five large regions of similarity with the enzymes from E. coli (lacZ, ebgA), Klebsiella pneumoniae (IacZ), and Lactobacillus bulgaricus are present in the PGal of C. thermosulfurogenes EM1 and that the putative active site residues (GAUGE’and Tyr503 in the E. coli IacZ-encoded BGal) are conserved (G1u389 and Tyr 429). Therefore, the thermostable

fiGa of C. thermosuljirogenes EM1 is more closely related to the enzyme of E. coli than to the likewise thermostable one of Bacillus stearothermophilus. The IacZ gene of C. thermosulfurogenes might be useful as a reporter gene in genetic systems where a low G + C content of the gene or thermostability of the enzyme is desirable.

INTRODUCTION

The /IGal from E. coli is well known due to extensive biochemical and genetic studies (Langridge, 1968; Beck-

Correspondenceto: Dr. H. Bahl, August-Universitat,

Institut

Grisebachstrasse

Tel. (49-551)393795;

Georg-

fur Mikrobiologie,

8, W-3400

Gottingen

(F.R.G.)

Fax (49-551)393793.

* Present

address:

Department

University

of Florida,

of Microbiology

3103 McCarty

and

Hall, Gainesville,

Cell

Science,

FL 3261 l-0144

(U.S.A.) Tel. (904) 392-5924;

Fax (904) 392-8479.

with and Zipser, 1970; Zabin and Fowler, 1980). However, in many other bacteria BGal is also induced during growth on lactose, e.g., in Streptococcus lactis, Staphylococcus aureus, Thermus strain T2, and Corynebacterium murisepti-

IPTG, isopropyl-@thiogalactopyranoside;

kb, kilobase

KP, K,HPO,/KHZPO,;

/3Gal from C. thermosulfuro-

/?-galactosidase(s);

aa, amino acid(s);

Ap, ampicillin;

ONPG(6P),

ONPG(6-phosphate);

acrylamide;

PAGE,

SDS, bp, base pair(s);

C., Clostridium; CAMP, cyclic AMP;

fiGal,

d, deoxyribo;

or 1000 bp;

genes EM 1; lucZpo,luc promoter-operator; LB, Luria-Bertani (medium); nucleotide(s); ONPG, o-nitrophenyl-fl-D-galactopyranoside; nt,

sodium

galactopyranoside; Abbreviations:

lucZ, gene encoding

a truncated

ORF, open reading

PA-gel electrophoresis;

dodecyl

sulfate;

XGal,

site;

5-bromo-4-chloro-3-indolyl-8_D-

[ 1,denotes plasmid-carrier

gene (3’ end).

frame; PA, poly-

RBS, ribosome-binding state;

’ (prime), denotes

14 CUM (Citti et al., 1965; Morse et al., 1968; Ulrich et al., 1972; Priyolkar et al., 1989). So far, the complete nt sequences of live different BGal-encoding genes have been published: IucZ (Kalnins et al., 1983) and ebgA (Stokes et al., 1985) from E. coli; IacZ from K. pneumoniue (Buvinger and Riley, 1985); from bgaB B. stearothermophilus (Hirata et al., 1986) and the unnamed gene of Lactobacillus bulguricus (Schmidt et al., 1989). As reported by Schmidt et al. (1989) there are seven regions of high similarity common to these PGal with the exception of the BGal from B. stearothermophilus. Therefore,

RESULTS

(a) Cloning of the 1acZ gene of Clostridium genes EM1

containing agar normally discriminates between E. coli clones with and without an insert in pUC18 (blue/white selection). However, one of the first 1000 transformants examined exhibited an increased fiGa activity, which could not be attributed to the r-complementation. A large blue halo was observed around the colony. The idea that the recombinant plasmid of the clone, designated pCT1. contained the IucZ gene of C. thermosuljiirogenes EM 1 was confirmed by the following results. pCT1 contained a 3.2-kb fragment of the chromosomal DNA from C. thermosdfurogenes EM 1, as shown by Southern hybridisation. Transfor-

encoded /3Gal) (Herrchen and Legler, 1984; Fowler and Smith, 1983) are conserved in all enzymes. The aim of this study was to investigate the thermostable fiGal of C. thermosulfurogenes EM 1, to clone the gene, determine the nt sequence, and compare the derived aa sequence with those of other BGal of different origin.

40

50

60

m

Temperature

5

I

6

thermosulfuro-

To establish a gene library of C. thermosdfurogenes EM 1~ its genomic DNA was partially digested with Sau3A and ligated into the BamHI site ofpUC18. After transformation into E. coli JM 105, clones were selected on LB agar plates, which contained Ap, XGal and IPTG. Plating on XGal-

it was possible that the enzymes from thermophilic organisms constitute a separate class of fiGal. The putative active-site residues (Glu4”’ and TyrSO’ in the E. coli lucZ-

30

AND DISCUSSION

80

[“Cl

8

9

Fig. I. Profiles of thermal stability (top) and optimal pH (bottom) of/?Gal activity in crude extracts ofB. coli JMlOS[pCTI] (o) and C. thermosulfurogenes EM 1 (0). BGal activity was spectrophotometrically determined using ONPG as substrate. The reaction mixture contained 980 pl Na phosphate buffer pH 7.0/20 pl 13.3 mM ONPGj20

~1 crude extract.

The amount

of o-nitrophenol

was determined

by measuring

absorbance

at 420 nm (a molar extinction

coefficient of s421, = 21.3 mM/cm was used); pH profile: PGal activity was assayed at various pH values at 60°C; thermal stability: samples were incubated for 30 min at the indicated temperature and /IGal activity was determined at pH 7.0 and 60°C. In crude extracts of E. coli the majority of E. co/i proteins was removed

by heat precipitation

(60°C.

30 min) before measurements.

15

pCTlO1

b

pCT102

b

1

+

(

+

pCTl03

kb Fig. 2. Restriction open bar represents Plasmids: XbaI-KpnI standard

pCTIO1, fragment procedures

map of the recombinant the Sau3A 3.15-kb

DNA fragment

S&I fragment

of pCT1 cloned (Sambrook

plasmid

of C. fhermosuljiwogenes

of pCTl

in pUC18.

of the IucZ gene of C. thermosulfurogenes

pCT1, subcloning cloned

in pUC18;

The fiGal activities

into the BamHI

EM1 integrated pCTiO2,

2.70-kb

were 0.6 units/mg

PsrI fragment

protein

EMI, and resulting

site of the multiple

of pCTl

cloned

( + ) or not detectable

/IGal activities.

cloning

in pUCI8;

The

site of pUCI8.

pCTIO3,

2.75-kb

(-)‘ DNA was manipuiated

by

et al., 1989).

mation of this plasmid into E. coli N99, a strain not capabie of cr-complementation, resulted in /?Gal activity of this strain. The temperature stability (up to 70°C) and pH optimum (7.0) of the enzyme expressed in E. cd corresponded to those of the C. thermo.~l~l~4rogenes EM 1 enzyme (Fig. 1). Finally, the enzyme from the clone migrated to the same position as the @Gal from C. thermosulfurogenes EM 1 as shown by gel electrophoresis under nondenaturing conditions and activity staining with XGal. As estimated from SDS-PAGE, the BGal size was 80 kDa (data not shown). These results indicated that the isolated clone contained the PGal-encoding gene (lacZ) of C. thermosulfurogenes EM 1. The restriction map of the clostridial DNA fragment of pCTl is depicted in Fig. 2. By subcloning of different restriction fragments it was shown that the lucZ gene was located on a 2.7-kb PsrI fragment in pCT102 (Fig. 2). (b) Nucleotide sequence of k.Z from Ci~st~jdiu~ themno-

su~f~~ogen~sEM1 The nt sequence of the 2.7-kb PstI fragment of pCT102 was determined (Fig. 3). One large ORF was found starting with ATG at nt 252 and ending with TAG at nt 2402. The ORF coded for a 716aa protein with a calcutated M, of 83 728. This is in good agreement with the size estimated by SDS-PAGE. To verify the reading frame of the la&Zgene, the enzyme expressed in E. coZiwas isolated and the N-terminal sequence determined (underlined in Fig. 3). Purifi-

cation of the enzyme was achieved by two simpte steps. Heat treatment (30 min at 60°C) precipitated the majority of E. coli proteins, whereas the thermostable PGal of C. thermosulfurogenes EM 1 remained soluble. Preparative gel electrophoresis of the soluble fraction followed by electro-eiution of the respective protein band, identified by activity staining towards XGal, yielded a purified enzyme (Fig. 4). The directly determined and the deduced aa sequences were identical and the protein synthesized in E. coli still contained Met as N-termina1 aa. Gradient-gel electrophoresis (6-25 % PA) and gel filtration on Superose 12HR of the purified enzyme revealed sizes of 172 and 175 kDa, respectively. These results suggested that the active flGa1 of C. thermosu~rogenes EM1 is composed of two identical subunits (Q). The la&? gene of C. thermosu@rogenes EM1 was expressed in E. cob independently from the presence of IPTG and from the orientation of the insert relative to the plasmid promoter. These results suggested that the DNA from C. thermosulfurogenes EM 1 contained promoter-like sequences from which transcription starts in E. coli. This could be confirmed by using the promoter test vector pKOl1 in E. coii N 100 (McKenney et al., 1981). The structural gene was preceded by a possible RBS and by putative -10 and -35 promoter regions for E. coli RNA polymerase, as indicated in Fig. 3. Further experiments are required to show that this region indeed functions as a promoter in

16 E. cofi. In addition, the functional promoter for C. thermosulfurogenes EM 1 RNA polymerase has to be identified. No typical Rho-independent transcription terminator was

1

found in the sequence upstream (251 bp) and downstream (320 bp) of the IacZ gene. Since these sequenced flanking regions are relatively short, it is possible that a terminator

CTGCAGCCGCTATATATCTTTTATGACAAGTTGGAATTCATATTTATGGCCTCTTATTATATTACAAAGTGATAAACAGAAGACGATGACTCTTATGTCATCTGCAT

-35 -10 120 ATTTCCAGATTATGGTGCAGTAATGGCTGGCTGCTATTGTTGTAGC~CTTTACCTATGAT~T~TATTCTTTGCGCTTC~GATATTTTGTGC~GGTTTGACAGGTGCAGT-CGTAG

24c 1 360 37

CATACAAATATAGAACTGCCATATAATTATTTTGATGAAAAAA TGTATCAGATTAAATCATGCTACAAATATCCACTTCCATTCTATATTCAT HisThrAsnIleGluLeuProTyrAsnTyrPheAspGluLysMetTyrGlnIleLysSerCysTyrLysTyrProLeuHisIleSerGluLysTyrA~gAspLysValIleTyrIleHis

480 17

TTTGAAGGAGTAATGGCGTATGCTCAAGTTTATTTGAATGGAGGATATACACCATTTGATATTAGAATTGATGAGGTTTATGACTGGAAGAAAGAA PheGluGlyValMetAlaTyrAlaGlnValTyrLeuAsnGlyLeuTyrIleGlyGluHisLysGlyGlyTyrThrProPheAspIleArgIleAspGluValTyrAspTrpLysLysGlu

600 117

TTGAATATGCTTACAGTGGTCGTTGATTCTACCG~G~GTGATATACCACC~GGAGGTCAGATAGATTATCTGACATATGGTGGTATTTATCGTG~GTGAGTTTAGGTATATAC LeuASnMetLeuThrValValVslAspSerThrGluArgSerAspIlePrO~lyIleTyr I GATGATGTATTTATTAAAAATATTAAAGTCGAAACACATGGAATATATATAATTTTGATTGTGCATTTAGAAAATTTAAATCATCAAAGTGGAAATGTCAAATTT AspASpValPheIleLySASnIleLysValGluThrHisGlyIleTyrAspAsnGluLysSerLeuAsnLeuIleValHisLeuGluAsnLeuAsnHisGlnSerGlyAsnValLysPhe

720 157 840 197 960 237 1080 277 1200 317 1320 357 1440 391 1560

AAGGTAAAAATRAATGATRGGC~G~GTTTTTTAT~G~TTT~CACATACTTAGATGCAGT~GATGTTTATTCTTTT~TATAG~TTTG~GATAT~GTTG LysValLySIleAsnASpLysAsnGlyLysGluValPheTyrLysGluPheAsnThrTyrLeuAspAlaValLysAspValTyrSerPheAsnIleGluAsnLeuLysAspIleLysLeu TGGGATGTGGATAACCCTAATTTATATGAGATTAAAGTGGGTATG~T~TAATTTTTCCGATG~TATGAC~T~TTTGGCTTTCGAG~GCTGTTTTT~CCTGATGGTTTC ~GluIleLysValGlyMetLysIleAsnAsnPheSerAspGluTyrAspAsnLysPhe~ II TACTTAAATGGCAGAAAATTAAAATTGAGAGGATTAAATAGGCATC~TCTTATCCGTATGTAGGTTATGCCATGCCTAGACGTGTTCAGG~GATGCTG~TTCT~TG~ ~SerTyrPrOTyrVslGlyTyrAlaMetP~oA~gA~gValGlnGluLysAspAlaGluIle~ 11a TTACATTTGAATATTGTTCGTACGTCGCATTATCCTC~TCT~CATTTTTT~AC~TGCGATG~CTT~GTTACTTGTATTTG~G~TTCCTGGGTGGC~TATATAGGG~T ~1l~P~0GlyT~pGl~~y~1kGlyA~~ III AGTGAGTGGAARAAA GTTGCAGAACAAAATTTAAGAGAAATGATTACRAGAGATTGGAATCATCATCAATTCTCAGGATGATGATGCTTTT SerGluTrpLysLysValAlaGluGlnAsnLeuArgGluMetIleThrArgAsp~GlvVa~GlnAspAspAspAlaPhe IV TATAARAATATGRATAAGATAGCTCATGAAATAGACCCTACGAGACAGACCGGAGGAGTCAGATATAT~C~TAGCAGTTTTCTTG~GATGTATATACATTT~TGATTTTATACAT TyrLysAsnMetAsnLysIleAlaHisGluIleAspProThrArgGlnThrGlyGlyValArgTyrIleThrAsnSerSerPheLeuGluAspValTyrThrPheAsnAspPheIleHis

437

GATGGGATTAATAAGCCATTGAGAAAACIlACAAGAAGTTACA~TCTTG~CAT~TGTACCTTATTTAGT~CAG~TAT~TGGTCATATGTATCC~CT~CGGTTTGAT~CG~ AspGlyIleAsnLysProLeuArgLysGlnGlnGluValThrGlyLeuGluHisAsnValProTyrLeuValThrGluTyrAsnGlyHisMetTyrProThrLysArgPheAspAsnGlu

1680 477

GAAAGGCAGATGGAACATTGCTTACGCCATTTAAGRATTCAAAATGCTTCTTATTTGGATGATAGTATTTCTGGTGCAATAGGTTGGTGTGCATTTGACTAT~TACACAT~GATTTT GluArgGlnMetGluHisCysLeuArgHisLeuArgIleGlnAsnAlaSerTyrLeuAspAspSerIleSerGlyAlaIleGlyTrpCysAlaPheAspTyrAsnThrHisLysAspPhe

1800 517 1920 557 2040 597 2160 637 2280 677 2400

2520

TTATTATATGAAACAGAAAGAGAATTTCATCTACAGATGATTATATAAGCTATATAATAATATGAAAAATAATCAGCTTGG~TTTATATTTTGGGAAGCGATTAAACAATAGAGA

2640

ATCTTTTAGTCATCTTATGATATATTGCCAAGAGGATATACTGCATGTGTTTATGAAGGCGATCCTCTAGAGTCGACCTGCAG

Fig. 3. Nucleotide of Sanger pCTlO2

sequence

of the PstI fragment

et al. (1977) with [s5S]dATP and the entire sequence

of pCT102

containing

and a T7 sequencing

of both strands

was determined

IucZ. Sequencing

kit of Pharmacia using successive

was performed

LKB, Freiburg, primers

F.R.G.

designed

using the dideoxy Single-stranded

according

chain-termination

templates

to the sequence

method

were prepared

already

determined.

from Only

the mRNA like (antisense) strand is shown. The deduced aa sequence is written below the nt sequence. The possible -35 and -10 sequences in the promoter region and the putative RB S sequence are marked. The N-terminal aa sequence (underlined) of the purified /?Gal of C. thermosuljiirogenes EM 1 (Fig. 4) expressed in E. coli, was determined by direct sequencing using a protein peptide sequencer 477A (Applied Biosystems, Foster City, CA). Detection was performed online with a phenylthiohydantoin analyzer. Before sequencing, the /IGal was subjected to SDS-PAGE (0.1% SDS/lo% PA) and blotted on a polyvinylidene difluoride membrane by using a Fast Blot apparatus (Biometra, Gdttingen, F.R.G.) (30 min, constant current [5 mA/cm’ of gel]). Three asterisks, stop codon. The aa in regions of high similarity in BGal of several bacteria (Schmidt et al., 1989) are also underlined and marked with Roman

numbers

(I-IV;

see Fig. 5). The accession

No. M57579

in the GenBank

database

has been assigned

to this nt sequence.

17 gene showed a strong bias towards codons using predominantly A and U as expected from the low C + C content.

kDa

92.5

66.2

45.0

-

31.0

Fig. 4. SDS-PACE (Laemrnli, 1970) of tht: p~r~~~a~~#n steps of j?Gal from C. thwmosulfurogenes EM1 expressed in E. coli. The proteins were separated in a 0.1 y0 SDS/IO% PA gel and visualized by staining with Cuomassie brilliant blue. Lanes: 1, crude extract of E. coli NQQ[pCTl]; 2, soluble fraction after heat precipitation (6WC, 30 min); 3, j?Cal protein electro-eluted out of a preparative gel ~no~denatn~ng, 7% PA, Trisjglytine buffer system pH 7.0) tier identification by activity staining with XGal; 4, marker proteins.

is located further down- and/or upstream. If this is not the case, the C. ~he~~s~~r5genes EM1 @Gal may be translated from a polycistronic mRNA. During growth of C. thermosuljiirogenes EM1 in a mineral medium, containing both glucose and lactose as carbon source, @+l synthesis is repressed as long as glucose is present in the medium (diauxic growth, data not shown). The consensus sequence for binding of the CAMP-catabolite activator protein complex of E. coli was not found upstream from facZ (de Crombrugghe et al., 1984). The iacZ gene of C. thermosulfurogenes EM1 had a G + C content of 31.5 mol”//,, compared with the value of 37 mol% reported for total genomic DNA of C. t/tern~o.sulfurogentIsg~~e~ EM1 (Madi and Antraniki~, 1989). Codon usage of the 1ac.Z

deduced aa sequence of the BGal of C. ~~er~~~~~~~genes EM 1 showed several legions similar to those of other jGa1 (Fig. 5). Schmidt et al. (1989) found seven common regions in fiGa from E. co!ofi(ebgA, lacZ) (Stokes et al.. 1985; Kalnins et al., 19833, L. ~~~~g~~~~~~ (Schmidt et af., 1989) and K. pneumoniue (Buvinger a,nd Riley, 1985). The first four regions could also be detected in the lncZ product of C. thermosulfurogenes EML. The similarity in the other three regions was, if at alf, low. However, an additional region of high similarity common to all five @Gal was identified between regions II and III, corresponding to aa 265-292 ofthe C. thermosuljiirogenes EM 1 @Gal. Region IV contained the likely active-site residue GIu~~’ (GAUGE’ in the E. co& &Z-encoded f36al; Herrchen and Legler, 1984). Also Tyrsa3 (E. cofi lucZ product), which is believed to be the proton-donating residue during hydrolysis of lactose (Fowler and Smith, 1983), might correspond to Tyr**” in the enzyme of C. ~herm~s~~r#genes EMI, since it is at a similar distance to the active-site GIu~*~ residue. Based on the high degree of similarities, it has been suggested by Schmidt et al. (1989) that flGa1 from E. co/i (lacZ, ebgA), L. b~~gar~~~sand K. ~~~e~~u~~~ehave been evolved from a common ancestor. The structure of the bgaB-encoded /?Gal from B. stearothermophilus was found to be quite different (Hirata et al., 1986; Schmidt et al., 1989). From our results it seems that thermostable fiGa do not constitute a separate class of fiGal. The thermostable enzyme of C. thermosulfurogenes EM 1 is more related to the enzymes of E. coli than to B. stearothermophilus enzymes [high similarity in the first four (five) conserved regions]. The fact that the last three conserved regions (V-VII) could not be found might be connected with the relatively small subunit size of the jGa1 from C. thermosdfurogenes EM1 (M, 83728). Interestingly, with respect to the size, the lucZ product is similar to the hga3 product of B. s~e~ro~~~ern~~~hi~~ (MT 78052). in addition, the thermostability of both enzymes is in the same range (up to 70°C; Hirata et al., 1984). Aliphatic bonding may contribute to the thermostability of these enzymes. It can be evaluated by the aliphatic index introduced by Ikai (1980), which is defined as the relative volume of a protein occupied by aliphatic side chains (Ala, Val, Leu, Ile) and which is significantly higher for many proteins of thermophilic than of mesophilic organism (Ikai, 1980; Hirata et al., 1986). The aliphatic index of the /?Gal of C. thermosu@kvgenes EM1 calculated according to Ikai (1980) was 85.4, similar to that of the enzyme from B. .~tear~~he~o~hi~us(84.5) and higher than that of pGa1 of E, coti (77.1). The

18 (1)

cts Eco

1acz 1acZ

PKGGQ 1-GL E D Q

Eco

ebgA Kpn 1acZ

iy_‘-IV E D Q 1” L E D Q

LbU

;WLEDQ ___ a

(11)

Cts 1acZ Eco 1acZ Eco ebgA Kpn 1acZ LbU

(IW

lacl Eco 1acZ Cts

ECO

ebgA

Kpn

1acZ

LbU

265 330 249 337 326

(111)

Cts Eco

1acZ 1acZ

Eco egbA Kpn 1acZ LbU

313 378 297 386 374

( IV)

Cts

1acZ

ECO

1acZ

Eco ebgA Kpn 1acZ

Lbu Fig. 5. Comparison

of aa sequences

(Eco, 1ucZ andebgA

products),

of five regions

K. pneumoniue

of high homology

of C. thermosulfurogenes EM1 (Cts) /3.3al to corresponding

(Kpn) and L. bulguricus (Lbu). The regions are numbered

et al. (1989). The aa are identified by the single-letter code and the positions of the aa in the enzymes solid lines (for three or more enzymes) or broken lines (identical aa in two enzymes).

(d) Use of the /?Gal-encoding gene of Clostridium thermosulfurogenes EM1 /IGal activity is detectable by simple methods. Therefore the IacZ gene of E. coli is being used as a reporter gene in a variety of applications (Sambrook et al, 1989). In cases where the special features of the j?Gal of C. thermosulfurogenes EM 1 (thermostability of the enzyme, low G + C content of the gene) are desirable, the 1acZ gene of this organism might be given preference over the ZacZ gene of E. coli. For example, C. acetobutylicum that has become a model organism for molecular biology work on nonpathogenic clostridia in recent years (for a review see Young et al., 1989) has a G + C content of 28 mol% (Cummins and Johnson, 1971), and several frequently used strains including the type strain have a phospho-fiGa but no /IGal activity (Yu et al., 1987; our unpublished results). The BGal of C. thermosulfurogenes EM1 showed no activity with ONPG(6P) as substrate. Therefore, the use of the 1acZ gene of C. thermosulfurogenes EM1 (G + C content of 31.5 mol%) as reporter gene in this organism is possible without the need to isolate lac- mutants, and any expres-

enzymes

(I), (II), (Ha), (III), and (IV) according are indicated.

sion problems due to limited species would be avoided.

Identical

residues

availability

from E. coli to Schmidt

are boxed with

of rare tRNA

(e) Conclusions (I ) The inducible /IGal of C. thermosulfurogenes EM 1 has a pH optimum of 7, and is stable up to 70°C. It has no activity with ONPG(6P) as substrate. (2) The 1acZ gene was cloned and expressed in E. coli, obviously by using its own promoter and RBS, and codes for a 716-aa protein with a calculated A4, of 83 728. (3) The active enzyme consists of two identical subunits. (4) The /IGal of C. thermosulfurogenes EM1 is more related to the corresponding enzymes of E. co/i, K. pneumoniae, and L. bulgaricus than to the thermostable /?Gal of B. stearothermophilus. (5) High aliphatic bonding may contribute to the thermostability of the j?Gal of C. thermosuffurogenes EM1 as indicated by a relatively high aliphatic index of 85.4, which was calculated according to Ikai (1980) (for the E. coli lacZ-encoded BGal the aliphatic index is 77.1). (6) The IacZ gene of C. thermosulfurogenes EM1 might

19 be useful as a reporter gene in genetic systems, where a low G + C content of the gene (i.e., for Clostridium species as C. perfringens and C. acetobutylicum) or thermostability of the enzyme is desirable.

Laemmli,

U.K.: Cleavage

Langridge,

J.: Genetic

structure Madi,

E. and

and enzymatic

G.: Identification producing

enzymes

Microbial.

53 (1987) 1661-1667.

K., Shimatake,

recognized

by Escherichia coli RNA polymerase.

Structural

T.S. (Eds.), Analysis

Gene

Laboratory, Buvinger,

D.: The Lactose

Cold Spring

W.E.

and

Harbor,

Riley,

M.:

Morse,

Nucleotide

J.E.,

Sandine,

W.E.

and

Elliker,

Streptococcus lactis. J. Bacterial. Cummins,

C.S. and Johnson,

of Klebsiella

sequence P.R.:

fi-Galactosidase

of

89 (1965) 937-942.

J.L.: Taxonomy

ofclostridia:

of Nucleic Acids. Elsevier, Amsterdam,

M., Nair, C.K.K. and Pradhan, of an inducible

F., Nicklen,

terminating 5463-5467.

acid-producing

J. Gen. Microbial.

P-galactosidase 625-635.

clostridia.

Fowler,

67 (197 I) 33-46.

activation.

A.V. and Smith,

P-galactosidases

Science

P.J.: The active

are

homologous.

Stokes,

224 (1984) 83 l-838. site regions

J.

protein:

Biol.

of IacZ and egb

Chem.

258

(1983)

Herrchen,

M. and Legler, G.: Identification

of an essential

group at the active site of IacZ /I-galactosidase Eur. J. Biochem. Hirata,

S. and Okada,

of fi-galactosidases

of two ,&galactosidases, Hirata,

H., Fukazawa,

fl-galactosidase

H.: Molecular

basis of isoenzyme

in Bacillus stearothermophilus: isolation

bgaA and bgaB. J. Bacterial.

T., Negoro,

S. and Okada,

160 (1984) 9-14. H.: Structure

of a

gene of Bacillus stearothermophilus. J. Bacterial.

166

(1986) 122-727. A.: Thermostability Biochem. Kalnins,

(Tokyo)

and aliphatic

index

of globular

proteins.

J.

A., Otto, K., Riither,

1acZ gene of E. coli. EMBO

U. and Miiller-Hill, J. 2 (1983) 593-597.

B.: Sequence

of the

T.: Molecular

Cloning.

Laboratory

A.R.: DNA sequencing Natl.

R.M., Requadt,

Acad.

C., Power,

sequence

Sci.

A

Press,

with chain-

USA

74 (1977)

S. and Mainzer,

S.E.:

of the Lactobacillus bulgaricus

H., Betts, P.W. and Hall, B.G.: Sequence

Escherichia coli: comparison

171 (1989)

of the ebgA gene of

with the 1acZ gene. Mol. Biol. Evol. 2

(1985) 469-477. G.A. and Temple, K.L.: Induction

zation of/I-galactosidase (1972) 691-698. the genetics 301-326.

in an extreme thermophile.

N.P. and Staudenbauer,

of the clostridia.

FEMS

tosidase

and phospho-/%galactosidase

and characteriJ. Bacterial.

W.L.: Recent Microbial.

Yu, P.-L., Smart, J.B. and Ennis, B.M.: Differential

Zabin,

I. and Fowler,

A.V.: /3-Galactosidase, transacetylase.

W.S. (Eds.), The Operon. Cold Spring Spring Harbor, NY, 1980, pp. 89-121.

1 IO

advances

in

Rev. 63 (1989)

induction

of /I-galac-

in the fermentation

permeate by Clostridium acerobufylicum. Appl. Microbial. 26 (1987) 254-257. tein, and thiogalactoside

88 (1980) 1895-1898.

and charac-

Corynebacterium

from

gene cloned in Escherichia coli. J. Bacterial.

Young, M., Minton,

138 (1984) 527-531.

H., Negoro,

formation

carboxylate

from Escherichia coli.

Proc.

and nucleotide

Ulrich, J.T., McFeters,

10204-10207.

W.: Metabolism

15 1 (1989) 49-53.

S. and Coulson,

B.F., Adams,

Expression

B., Busby, S. and But, H.: Cyclic AMP receptor

J.G. Vol. 2.

198 1, pp.

D.S.: Purification

/?-galactosidase

E.F. and Maniatis,

inhibitors.

in Closfridium bufyricum and other butyric

role in transcription

Ikai,

J., Fritsch,

tion and DNA homologies de Crombrugghe,

and Analysis,

Laboratory Manual, 2nd ed. Cold Spring Harbor Cold Spring Harbor, NY, 1989.

Schmidt,

wall composi-

Amplification

signals

In: Chirikjian,

M.L., Hill, K.L., Egan, J.B. and Hengstenberg,

Sanger,

163 (1985) 850-857.

U., Brady, C. and

and terminator

of lactose by Staphylococcus aureus and its genetic basis. J. Bacterial. 95 (1968) 2270-2274.

Sambrook,

Cold Spring Harbor

NY, 1970.

pneumoniae lac genes. J. Bacterial. Citti,

Operon.

Appl. Environ.

383-415.

terization

J.R. and Zipser,

and

G.: Thermostable

H., Court, D., Schmeissner,

murisepticum. Arch. Microbial. Beckwith,

K. and Gottschalk,

M.: A system to study promoter

and Papas,

degrading

z-amylase

30 (1989) 422-425.

Rosenberg,

Priyolkar, REFERENCES

of a starch

from a new Clostridium isolate.

amylolytic

of

relating to the tertiary

thermostable

Biotechnol.

G., Ohmiya,

the assembly

96 (1968) 171 I-1717.

Antranikian,

Madi, E., Antranikian,

McKenney,

experiments

J. Bacterial.

Appl. Microbial.

during

227 (1970) 680-685.

thermophile

pullulanase.

This work was supported by a grant of the ‘Bundesministerium fur Forschung und Technologie’. We thank C. Marck, CEN-Saclay, Gif-Sur-Yvette (France) for a copy of the DNA Strider program. We are obliged to K. Herwig for photography, B. Schmidt for determining the N-terminal aa sequence and G. Gottschalk for generous support (all of them from Universitat Gottingen).

proteins

T4. Nature

of /3-galactosidase.

anaerobic

ACKNOWLEDGEMENTS

of structural

the head of bacteriophage

the lactose

of whey Biotechnol.

permease

pro-

In: Miller, J. and Reznikoff, Harbor

Laboratory,

Cold