Journal o/Biorechnology, 4 (1986) 181-187 Elsevier

181

JBT 00208

Isolation and preliminary characterization of plasrnids in Bacillus badius

Anu Jalanko i, Matti Leisola 2** and Veli Kauppinen 3

I Recombinant DNA Loboratory University of Helsinki, 00380 Helsinki, Finland,

’ Department of Biotechnology, Swiss Federal Institute of Technology, 8093 Ziirich, Switzerland, and ’ Biotechnical Luborarory Technical Research Cenrre of Finland, 02150 Espoo, Finland

(Received 3 March 1986; accepted 10 March 1986)

Summary

The presence of urease and NADPH-specific glutamate dehydrogenase (GDH) coding plasmid in Bacillus badius was suggested by the loss of enzyme activities with a mitomycin C treatment and by the presence of 3 plasmids in urease-active strains compared to 2 plasmids in urease-negative strains. Furthermore two-dimen- sional protein gel electrophoresis showed that the urease-positive strains had some additional proteins compared to urease-negative strains. The two common plasmids had a size of 14.7 kb and 5.9 kb. The plasmid that was missing in the urease-nega- tive strains had a size of 44.0 kb. The plasmids were purified and restriction map for AuaI, BamHI and EcoRI was constructed for each plasmid. Hybridization tests showed that all three plasmids in Bacillus badius were independent entities.

plasmids, Bacillus badius, urease, NADPH-dependent glutamate dehydrogenase

Introduction

The microbial flora in uremic human gut is able to adapt itself to utilize the endproducts of nitrogen metabolism normally secreted through kidneys, although no significant qualitative or quantitative differences between the uremic and healthy human fecal microbial flora have been found (Suominen et al., 1982).

* To whom all correspondence should be addressed. Abbreviations: LGT, low gelling temperature (agarose); SDS, sodium dodecyl sulphate; bp, base pairs.

0168-1656/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

182

Uremic symptoms have been treated using lyophilized soil bacteria active for degrading substances like urea, creatinine and uric acid (Setala et al., 1973; Setal& 1978). The promising results led to a further study (Kauppinen, 1982) to improve the degradation activities of specific micro-organisms by ordinary screening tech- niques. In these studies urease and NADPH-specific glutamate dehydrogenase (GDH) activities in Bacillus badius showed a similarly discontinuous distribution pattern after cloning the organism into 50 single cell cultures with 8% of the cultures being urease-negative. The results implied a common plasmid coding for these enzymes.

The present study was undertaken to find out whether B. badius contains plasmids, to preliminary characterize the possible plasmids and to clarify somewhat more their role in coding for urease and GDH activities.

Materials and Methods

Bacillus badius strains 14/77, 16/77, 28/77 and 29/77 were isolated by Kaup- pinen (1982). E. coli V517 strain containing 8 plasmids of known molecular weight (Macrina et al., 1978) was received from Dr. J. Knowles (Biotechnical laboratory, State Research Centre of Finland, SF 02150 Espoo 15, Finland) and was used to obtain molecular weight standards.

B. badius was grown in a medium containing 1% beef extract, 2% glucose and a mineral salt mixture (Kaltwasser, 1971) for 11 h at 37°C on a gyratory shaker (240 ‘pm). The cells from 2 1 cultures were harvested and plasmid DNA isolated by the SDS-NaCI method (Guerry et al., 1973) followed by polyethylene glycol precipita- tion (Humphreys et al., 1975). CsCl-EtBr dye buoyant-density ultracentrifugation was carried out according to Radloff et al. (1967). The plasmid DNA band from density gradient was fractionated into discrete bands by preparative electrophoresis on 0.75% LGT agarose gel. Plasmid DNA bands were cut from the gel and the DNA eluted out from the agarose by melting the gel with equal volume of TE-buffer (10 mM Tris, pH 8.0; 1 mM EDTA) at 65°C. The aqueous phase was re-extracted with phenol at room temperature, residual phenol removed with diethyl ether and residual ether by gassing the solution with nitrogen. The plasmid DNA was then precipitated with ethanol and used for restriction enzyme analysis or hybridization experiments.

Plasmid DNA of known molecular weights was isolated from E. cofi V517 by the method of Clewell and Helinski (1969).

Urease and NADPH-specific glutamate dehydrogenase (GDH) activities were measured from cell free extracts (Kauppinen, 1982) and expressed as nM NH, formed per mg dry cells in 1 min. Two-dimensional protein electrophoresis of total cell proteins (O’Farrell, 1974) was carried out using the same extracts after dialysis. In the first dimension following ampholytes (Biorad) were used: 3/10 : 3/5 : 5/7 : 6/8 = 2 : 3 : 5 : 2. For the second dimension 9% polyacrylamide gels were used.

183

Mitomycin C treatment for plasmid curing experiments was carried out as described by Chakrabarty (1972).

Reaction mixtures for restriction endonuclease digestion contained 0.5 pg of plasmid DNA in a final volume of 10 ~1. The plasmids were digested for 2 h at 37°C with 0.5 U (New England Biolabs enzymes) or 2 U (Bethesda Research Laboratories enzymes) of enzyme in 10 mM Tris buffer, pH 7.6, containing 50 mM NaCl, 10 mM MgCl, and 1 mM dithiothreitol (Sigma).

Plasmid DNA and DNA fragments were resolved either by horizontal or vertical 0.6-1.0% agarose gel electrophoresis (Hayward and Smith, 1972) at constant voltage of 20 V (1 V cm-‘) overnight at room temperature. Gels were stained with ethidium bromide (1 pg ml-‘) and photographed under UV light on Polaroid 665 or 667 film using Kodak Wratten No. 23A filter.

Estimation of the molecular weights of plasmids was performed using plasmids from E. coli V517 as standards. The molecular weights of plasmid fragments were determined with the EcoRI-Hind111 digested fragments of lambda phage DNA (Bethesda Research Laboratories) as standards (Szybalski and Szybalski, 1979).

Hybridization tests were done as follows: DNA from 0.6% agarose gel was transferred to Schleicher & Schiill BA 85 nitrocellulose filter, washed, heated and prehybridized according to the procedure of Southern (1975). Plasmid DNA probes were prepared by labeling with [a- ‘*P]dCTP (Amersham Searle Corporation) according to the nick translation procedure (Rigby et al., 1977). Hybridizations were carried out in sealed plastic bags at 37°C for 36-48 h in a total volume of 1 ml. For hybridization 1 x lo6 cpm of labeled probe was used. Filters were washed according to the Southern protocol, dried and incubated at -70°C for 24 h with an intensifying screen for autoradiography.

Results and Discussion

In the presence of 5 pg ml-’ mitomycin C 38% of the urease positive cultures lost their activity completely while 62% retained their activity (Table 1). At the same time also the GDH activity was affected. However it was not totally lost together with the urease activity but reduced to 50% of the original value. When the urease activity was not changed as a result of mitomycin C treatment then also GDH activity remained constant. This result implied a common plasmid coding for both enzymes as well as an additional chromosomal coding for GDH.

In order to confirm the presence and the total number of plasmids in the different B. b&us strains a preparative scale isolation of plasmids was carried out using CsCl-gradient centrifugation technique. After centrifugation the cccDNA band was fractionated by electrophoresis in 0.6% agarose gel. Urease active B. bad& strains 16/77 and 29/77 contained three plasmids of the size 44 kb, 14.7 kb and 5.9 kb while the urease-negative strains 14/77 and 28/77 each contained two plasmids of the size 14.7 kb and 5.9 kb.

The three plasmids from B. badius 29/77 and the two from 28/77 were purified by preparative gel electrophoresis and subjected to restriction enzyme analysis with

184

TABLE 1

EFFECT OF MITOMYCIN C ON UREASE AND NADPH DEPENDENT GLUTAMATE DEHY- DROGENASE (NADPH-GDH) ACTIVITIES IN BACILLUS BADIUS

Activities are expressed as nM NH, formed per mg dry cells in 1 min.

Mitomycin C Proportion of cultures (S)

Urease NADPH-GDH activity activity

1OOn 190 80

+ 62 190 80 38 0 40

n In 3% of the cultures with urease activity the NADPH-GDH activity was lowered to 55 even without mitomycin C treatment.

TABLE 2

NUMBER OF RESTRICTION ENZYME CLEAVAGE SITES OF BACILLUS BADIUS PLASMIDS FOR SEVEN RESTRICTION ENZYMES

Enzyme Number of cleavage sites

44 kb plasmid 14.7 kb plasmid 5.9 kb plasmid

ArmI BumHI EcoRI BglII Hind111 HincII Sal1

2 0 1 1 1 3 1 5 0 4 6 0

>5 4 3 5 3 2

=-5 >5 1

kilobases

Fig. 1. Restriction enzyme cleavage site maps for Bacillus badius plasmids (a) 44 kb plasmid; (b) 14.7 kb plasmid; (c) 5.9 kb plasmid.

185

44 kb ccc

14.7 kb oc

a b

Fig. 2. Southern blot hybridization of Buci/lus bad& plasmids. (a) Samples (1 and 2) containing the 44 kb, 14.7 kb (ccc- and oc-forms) and 5.9 kb (ccc- and oc-forms) plasmids, (b) the same samples after hybridization (1.2). Sample 1 was hybridized with [a- 3ZP]dCTP-labeled 14.7 kb plasmid and sample 2 with [ a-‘*P]dCTP-labeled 5.9 kb plasmid.

seven enzymes. The digestion patterns for the 14.7 kb and 5.9 kb plasmids from both strains were identical for each size class. The number of fragments generated from the three plasmids of the strain 29/77 is summarized in Table 2.

Comparison of digestion patterns on 0.6% and 1.0% agarose gels revealed that each of the three plasmids produced a unique restriction pattern for each enzyme used. The plasmids did not share any obvious common restriction fragments.’ Double digestions were performed to construct a map of the restriction sites of enzymes AuaI, BumHI and EcoRI (Fig. 1). The sum of the fragment sizes obtained from a given digestion was always close to the original molecular weight of each plasmid indicating that all major fragments were detected.

Purified samples (300 ng) of the 14.7 kb and 5.9 kb plasmids were nick translated with [cY-~‘P]~CTP. Both of these radioactive probes were separately hybridized with a nitrocellulose filter containing all the three plasmids transferred from gels by the Southern blot method. Autoradiographs of the nitrocellulose filters revealed that the 14.7 kb and 5.9 kb plasmids only hybridized with themselves (Fig. 2). Neither of them hybridized with the 44 kb plasmid. Hybridization experiments thus confirmed that all three were individual plasmids.

Two-dimensional protein electrophoresis of the total cell proteins of the urease positive and urease negative strains gave almost an identical result. However the urease positive strains had some proteins that were missing in the negative strains as indicated in Fig. 3. This is a further confirmation that the 44 kb plasmid is involved in synthesis of intracellular proteins.

The presence of urease and GDH genes in 44 kb plasmid will eventually be proved by transformation. The variation in these enzyme activities as reported by Kauppinen (1982) seems to be dependent not only on the copy number since cultures with high as well as low activities gave the same amount of 44 kb plasmid DNA. The study of these plasmids may thus give new ideas of the mechanism of enzyme regulation in Bacillu.s. Furthermore, the characterization of these plasmids

186

+

IEF -

i S +

-- f

I

l . .

.

v . l

. .

. - .

Fig. 3. Two-dimensional electrophoresis of the intracellular proteins of urease-positive ( +) and urease- negative (-) strains of Bucilluc budius. 500 pg of protein was applied.

is of interest since the function of only very few naturally occurring plasmids in Bacillus is known (Gryczan, 1982). In our preliminary tests we have not been able to find any specific function for the 14.7 kb and 5.9 kb plasmids.

187

References

Chakrabarty, A.M. (1972) Genetic basis of the biodegradation of salicylate in Pseudomonm. J. Bacterial. 112, 815-823.

Clewell, B.B. and Helinski, D.R. (1969) Supercoiled circular DNA protein complex in Escherichh co/i: puritication and induced conversion to an open circular DNA form. Proc. Natl. Acad. Sci. U.S.A. 62, 1159-1166.

Gryczan, T.J. (1982) Molecular cloning in Bacillus subtilis. In: The Molecular Biology of the Bacilli (Dubnau. D.A., Ed.), pp. 307-329. Academic Press, New York.

Guerry, P., Le Blanc, D.J. and Falkow, S. (1973) General method for the isolation of plasmid deoxyribonucleic acid. J. Bacterial. 116, 1064-1066.

Hayward. G.S. and Smith, M.G. (1972) The chromosome of bacteriophage TS. I. Analysis of the single-stranded DNA fragments by agarose gel electrophoresis. J. Mol. Biol. 63, 383-395.

Humphreys. G.D., Willshaw. G.A. and Anderson, ES. (1975) A simple method for the preparation of large quantities of pure plasmid DNA. B&him. Biophys. Acta 383, 457-463.

Kaltwasser. H. (1971) Studies on the physiology of Eacilhu fmridiosuc. J. Bacterial. 107, 780-786. Kauppinen, V. (1982) Variation of some enzyme activities involved in the utilization of urea, creatinine

and uric acid in bacteria. Acta Pharm. Fenn. 91, 87-95. Macrina, F.L., Kopecko, D.J., Jones, K.R., Ayers, D.J. and McCoven, S.M. (1978) A multiple plasmid-

containing Escherichia co/i strain: convenient source of size reference plasmid molecules. Plasmid 1, 417-420.

O’Farrell, P.H. (1974) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007-4021.

Radloff, R.. Bauer, W. and Vinograd, J. (1967) A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57, 1514-1520.

Rigby, P.W.. Dieckmann, M.. Rhodes, C. and Berg, P. (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113. 237-251.

Set&la, K. (1978) Bacterial enzymes in uremia management. Kidney Int. 13, Suppl. 8, 194. Setill& K., Heinonen. H. and Schreck-Purola, I. (1973) Ingestion of lyophilized soil bacteria for

alleviation of uremic symptoms. IRCS Med. Sci. (73-10) 10-19-7, 35. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel

electrophoresis. J. Mol. Biol. 98, 503-517. Suominen, M., Schneitz. C. and Kauppinen. V. (1982) Characteristic bacteria in the uremic and healthy

human gut. Proc. XIIIth Inc. Congr. Microbial. 8-13 August 1982, Boston, pp. 10-18. Szybalski. E.H. and Szybalski. W. (1979) A comprehensive molecular map of bacteriophage lambda.

Gene 7. 217-270.