Site-specific restriction endonucleases in Bacillus licheniformis

ELSEVIER

FEMS Microbiology

Letters 132 (1995) 285-289

Site-specific restriction endonucleases in Bacillus licheniformis Carlo Parini

*, M.

Received 2 I June 1995: revised

Grazia Fortina

I3 August 1995: accepted 25 August I995

Abstract We systematically studied site-specific restriction endonucleases in Bacillus lichenijormis strains and detected endonuclease activity in 25 of 217 strains tested. Three different activities were obtained. One of these activities detected in 21 strains was the most representative within the species and produced a banding pattern, after digestion of A DNA, identical to that seen with ChI. Two other strains isolated from soil samples from China and USA were found to produce a DNA-cleaving enzyme with the same recognition sequence as BsaI. One producer strain, isolated from a Peruvian soil sample, showed to possess a mixture of two isoschizomers, C/a1 and BsaI. Finally, one strain produced an endonuclease activity, not previously described in B. lichen(fi~rmis, that showed the same recognition sites as Bsu361. Kewordst

Bacillus

lichen~fk-nis:

Type II restriction

endonucleases;

1. Introduction Bucillus lichen@mis strains are safely used in the fermentation industry to produce a variety of products such as bacitracin, penicillinase, alkaline phosphatase, cr-amylase, protease and surfactant [ l41. These products are widely used in such industries as applied chemistry, tannery, food and feed. B. lichenijbrmis is therefore an attractive host for the expression of cloned gene products on an industrial scale. Several recent reports describe cloning and expression of industrially relevant proteins in B. licheniformis

[5-81.

Genetic engineering of bacterial species has been widely used in basic research and for industrial

i Corresponding 7063 8625. Federation SSDI 0378-

author. Tel.: + 39 (2) 236 7285; Fax: + 39 (2)

of European Microbiological 1097(95)00328-2

Societies

Bsul: C/al; Bsu361 isoschizomers

applications. However, transformation can be compromised by endogenous restriction nuclease systems within particular strains. Therefore, one objective vital for future research is the establishment of a stable DNA transfer system between bacteria with a well-studied genetic system. To improve transformation efficiency, it is desirable to determine which strains of a bacterial species have restriction endonuclease activity. A comprehensive survey of strains might lead to the discovery of novel restriction endonucleases or isoschizomers of known enzymes from more desirable sources. The distribution of restriction endonucleases has been systematically studied in the genus Bacillus and many species were reported to possess sitespecific endonucleases. However few reports concerning restriction enzyme production by B. licheniformis are known [9]. The first communication was from our laboratory which isolated an isoschizomer

of HueJII, named BliJ [IO]. Subsequently, other researchers reported the purification of three isoschizomers of C/a1 (B/i41 I, B/iX6J, B1I’RJ) and one of Eco3 I I (Bli49J) [9]. Since an investigation is in progress in this laboratory on the geographic distribution of the B. lichen~f~wmis species, it seemed interesting to study the presence and characteristics of restriction endonucleases in a large number of strains isolated from different geographic areas. The aim of the research presented here was to adequately describe the principal site-specific endonucleases present in B. 1ichetGfiwmi.s species and to point out if one of these is the most representative of the species.

2. Materials

analysis of endonuclease activity. Protocol for testing crude extract for endonuclease was carried out according to Poch and Somkuti [I I]. Restriction endonucleases were isolated from 2 I of a 8-h culture of B. lichen~fbrmis. The cells were harvested by centrifugation, disrupted by two passages through a French pressure cell at I2 000 lb in ‘, and the lysates were purified by essentially the same procedure as previously described [IO]. We were able to remove the majority of contaminating proteins. The enzymes were purified with a purification factor of 90. 2.3. ASSUJ ,fiw endonuclruse The protocol amount of I pg reaction

and methods

2.1. Buctericrl strains and culture media We tested 217 strains of B. lichen@mis available from the culture collection of our laboratory. All strains were originally isolated from samples of soil coming from different geographic areas (Table I>. The strains were cultivated in LB media at 42°C. 2.2. Engvne isolation and pur@%_ation Cells harvested from IO ml of stationary-phase culture were employed

an early for rapid

actil.it?

enzymes were assayed by using standard [ 121. We defined I U of endonuclease as the of enzyme required to produce a limit digest of A phage DNA in 1 h at 37°C in a 50-p.1 mixture.

2.4. Enzyme chmructeri:ation In order to determine optimal salt concentrations, assays were run with SURE/Cut buffer for restriction enzymes A, B, H, L. M (Boehringer, Mannheim) with 0.5 pug of A phage DNA and 1 U of enzyme. The definition of enzyme activity was at 37°C. The recognition sequence of purified endonucleases was identified using digestions on A phage, pBR322,

Table I Origin of the 2 17 strains of B. /ichm~i~mis Number of strains

Origin

tested and restriction endonuclease activity detected Positive strains

Origin

Number of stains

Positive strains

isolated

isolated China

46

I

Czech Republic

3

0

Italy

16

0

Auhtria

I

0

6

0

Tunisia

3

0

USA

26

5

Wales

1

0

Egypt

Canada

47

6

Australia

IS

I

Madagascar

I

0

Argentina

3

0

Island of Bali

3

0

Peru

2

I

Island of Nios

5

0

Ecuador

4

2

Island

of Celihes

3

0

Galapagos

5

3

Island of Java

3

0

France

2

0

Island of Sumatra Germany

I 3

0 0

ISrdcl Japan

I

0

IO

s

Croatia

2

0

Norway

3

0

Ruanda

4

I

M13mp18RF and pUC18 DNAs and by comparison with DNA digests of characterized restriction endonucleases.

The purity of final endonuclease respect to ‘non-specific nucleases’ ‘over-digestion’ tests. 2.6. Other

preparations with was evaluated by

methods

Protein concentrations were determined by the method of Bradford [ 131. Bovine serum albumin was used as standard.

3. Results and discussion

Restriction endonucleases have been screened in 2 17 B. lichen~fornzis strains. Four different activities were isolated and purified. According to the conventional nomenclature [I41 they should be termed BliS8.51. Bli64.31, Bli 16.11 and Bli.57.61. For all endonuclease preparations, maximum activity in SURE/Cut buffer H at 37°C was observed. The enzymes were stable and no significant decrease in the cleavage activity was observed during storage at - 20°C for several months. When the final enzyme preparations were subjected to over digestion tests of up to 30 U, non-specific cleavage products were observed even after 72 h of incubation. An attempt to identify the recognition sequences of the purified endonucleases was carried out using A phage. pBR322. M I3mpl8RF. and pUC 18 as substrate DNAs. The BliS8.51 endonuclease restricted A DNA to a pattern which is identical to that created by ClnI. This suggested that BIiS8.51 and CIaI must be considered isoschizomers. This assumption was confirmed by the restriction patterns obtained from parallel digestions of A phage DNA by Bli58.51 and C/al. The patterns were identical in each case (data not shown). Of the four isolated endonuclease activities. Bli58.51 seemed to be the most representative in the R. lic~her~jfi~rmis species. The data presented in

this paper show that 21 of the 25 positive strains produce an isoschizomer of ClaI. These findings appear to be in agreement with the literature [9]: in fact, three out of the five described endonucleases obtained from this species, namely BIi42I. B/i861 and BliRI are considered isoschizomers of Clul. The restriction endonuclease named Bli64.31 showed two recognition sites on A DNA: linearized M 13mpl8RF DNA; and did not cut pBR322 and pUCl8 DNA. The only restriction enzyme which has two restriction sites on A DNA and showed the same site-specificities on the other DNAs mentioned above appeared to be Bsu361. A double digest of Bli64.31 and Bsrr361 on A EroRI and A Hirrdlll digests confirmed that Bli64.31 was an isoschizomer of B.~u36 I. It is noticeable that, up to now, restriction enzymes with the same recognition sites have also been found in other microorganisms but not in B. /ic~hrr~ifi,mis [9]. This is the first communication on the production of a isoschizomer of Bsu361 by B. licher~$ifi,rnzis species. The restriction fragments after digestion of A phage DNA with Blil6. I1 seemed to show a similar pattern to Asc.1 or B.scrI. Additional support of these conclusions was obtained when A EcoRI digest, A HirrdIII digest. pBR322. Ml3mpl8RF and pUC 18 DNA were cleaved by Blil6. II. Asc~l and BsuI. A.sc.1. B.vaI and Blil6. II cleaved A DNA generating three fragments of similar molecular mass. AscI was unable to cleave pBR322, M l3mp 18RF and pUC 18. whereas Bwl and Blil6.11 showed one recognition cite in pBR322 and pUC I8 and none in M l3mp I8RF. Therefore Bli16.1 I appeared to be an isoschizomer of Bscrl. To confirm this hypothesis. parallel digestions of A-EcoRI and A-Hind111 digests by Bsal and Blil6. I I were carried out (data not shown). The patterns were identical, thus we believe that Bli 16.11 is an isoschizomer of Bsal. BliS7.61 apparently did not reveal any similarity with characterized enzymes that showed the same number of cleavage-sites in the tested substrate DNAs. In A phage DNA, this restriction enzyme recognized sixteen sites and produced I7 fragments. The size of the molecular mass of A fragments were near those predicted from A-Clal digests, with the exception of fragments of I I 385 and 1804 bp that were not produced by Bli57.61 (Fig. I). Circular pBR.322 and M l3mp l8RF DNA were cleaved twice:

288

C. Parini,

M.G. Fortina /FEMS

Microbio/og,v

plasrnid pUC18 was cleaved once (Fig. 2, lanes 4, 7, 10 and 13). The location of the unique Bli57.61 site in pUC I8 DNA was determined by double digests of BIi57.61 and either BamHI, EcoRI or ScaI. The results indicate that the Bli.57.61 site was located near the BsaI site. The mobilities of fragments of pBR322 obtained after digestions with Bli57.61 and BamHI or EcoRI, ScuI, Ar~1, and PMII led us to conclude that sites Bli57.61 were nearby or identical to sites BsaI and CluI (data not shown). To confirm these data, pBR322 was digested with Bli57.61, BsaI-Clu I or both. As shown in Fig. 2 (lanes 7-9), the restriction patterns were identical and the size of molecular mass fragments (3450 and 980 bp) were near those predicted (3410 and 951 bp) from the nucleotide sequence. Furthermore, the restriction patterns obtained from parallel digestions of standard substrate DNAs by BIi57.61, BsuI-C/a1 or Bli57.61 + BsaI-CluI were identical in all cases (Fig. 2). On the basis of these results, the endonuclease activity produced by strain 57.6, previously named Bli57.61. may be considered a mixture of two isoschizomers of CluI and BsaI.

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Fig. 2. Determination of the approximate sizes of fragments and comparison of the activities of Bli57.61 and BsaI-Clnl. Lanes l-3, B.stEII, Pstl and C/r11 fragments of A DNA as size markers respectively: lanes 4-6, Bli57.61. B.wI-C/u]. B/i57.61+ BscrlCltrI digests of A DNA; lanes 7-9. Bli57.61, BstrI-C/nI, Bli57.61 + BsaI-C/c/I digests of pBR322: lanes 10-12: RliS7.61. BsrrIC/t/I, B/i57.61+ Bw-C/n1 digests of Ml3mpl8RF; lanes ISIS, 811’57.61. &L/I-C/r/I. R/i57.61+ BsnI- C/u1 digests of pUC 18. Note that the samples were incompletely digested in lanes I3- 15. arrow indicates undigested pUC IX DNA.

These findings show that different endonuclease activities in B. lichen~fbnis are present and among these are ones not yet described. This indicates that B. lichen~formis strains may be considered an interesting source of this class of enzymes and spurs the investigation of this microbial species with increased interest.

Acknowledgements This work was supported by the Research Council Italy. Special Project RAISA, Sub-project 4, Paper No. 2349.

Fig. I. To verify that BIiS7.61 is a mixture of two isoschizomers of B.wI and C/al, A DNA was digested with: Bli57.61, lane 3: BsaI + C/uI, lane 4; C/(11, lane 5. Lane I, marker, DNA size in bp for the fragments are indicated in the left margin; lane 2. Hind111 fragments of A DNA. The arrows in the right margin show two bands of A-C/n1 digests cleaved by RstrI.

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